Methods of detecting trichomonas vaginalis

ABSTRACT

Compositions and methods for detecting  Trichomonas vaginalis  are provided.

1. FIELD OF THE INVENTION

Compositions and methods for detecting Trichomonas vaginalis areprovided.

2. BACKGROUND

The protozoan Trichomonas vaginalis is responsible for trichomoniasis,which is a common sexually transmitted infection that can infect bothmen and women. There are 7.4 million cases of trichomoniasis annually inthe United States. Trichomoniasis infections can be symptomatic orasymptomatic. See, e.g., Ginocchio et al., J. Clin. Microbiol. 2012, 50:2601-2608. In women, trichomoniasis is one of a range of conditions thatcomprise vaginal discharge. See, e.g., Centers for Disease Control andPrevention (CDC). CDC fact sheet: trichomoniasis. 2010.www.cdc.gov/std/trichomonas/STDFact-Trichomoniasis.htm. Symptoms infemales can include itching, burning, redness, or soreness of thegenitals, unusual odor, discomfort with urination, or a thin clear,white, yellow, or green discharge. See id. In men, trichomoniasis maycause non-gonococcal urethritis (NGU). Symptoms in males can includeitching or burning inside the penis, burning after ejaculation orurination, or penile discharge. See, e.g, Workowski et al., Centers forDisease Control and Prevention. Sexually transmitted disease treatmentguidelines, 2010. MMWR 2010; 59 (RR-12):1-110; Centers for DiseaseControl and Prevention. Biosafety in Microbiological and Biomedicallaboratories. www.cdc.gov/biosafety/publications/.

Improved methods for detection of Trichomonas vaginalis (TV) are needed.In particular, a highly specific, accurate, and sensitive urine- orswab-based diagnostic test is needed.

3. SUMMARY

In some embodiments, methods of detecting the presence or absence ofTrichomonas vaginalis (TV) in a sample from a subject are provided. Insome embodiments, methods of determining whether a subject has aTrichomonas vaginalis (TV) infection are provided. In some embodiments,the methods comprise detecting the presence or absence of the TV 40Sribosomal protein (Tv40Srp) gene or RNA in a sample from the subject.

In some embodiments, the subject has not previously been treated for TVinfection. In some embodiments, the subject has previously been treatedfor TV infection. In some embodiments, the previous treatment comprisedone or more doses of metronidazole or tinidazole. In some embodiments,the subject does not have any symptoms of TV infection. In someembodiments, the subject has one or more symptoms of TV infection. Insome embodiments, the subject has one or more symptoms selected fromvaginitis, urethritis, and cervicitis. In some embodiments, the subjectis female and has one or more symptoms selected from itching, burning,redness, and/or soreness of the genitals; unusual odor of the genitals;discomfort with urination; and a thin clear, white, yellow, or greendischarge. In some embodiments, the subject is pregnant. In someembodiments, the subject is male and has one or more symptoms selectedfrom itching and/or burning inside the penis; burning after ejaculationand/or urination; and penile discharge.

In some embodiments, the method comprises detecting an endogenouscontrol. In some embodiments, the endogenous control is a sampleadequacy control. In some embodiments, the endogenous control is asingle-copy human gene. In some embodiments, the endogenous control isselected from HMBS, GAPDH, beta actin, and beta globin.

In some embodiments, the method comprises detecting an exogenouscontrol. In some embodiments, the exogenous control is a sampleprocessing control. In some embodiments, the exogenous control comprisesa DNA sequence that is not expected to be present in the sample. In someembodiments, the exogenous control is a bacterial gene.

In some embodiments, the method comprises PCR. In some embodiments, themethod comprises quantitative PCR. In some embodiments, the PCR reactiontakes less than 2 hours, less than 1 hour, or less than 30 minutes froman initial denaturation step through a final extension step.

In some embodiments, the the TV 40S ribosomal protein (Tv40Srp) genecomprises the sequence of SEQ ID NO: 4. In some embodiments, the methodcomprises contacting nucleic acids from the sample with a first primerpair for detecting the TV 40S ribosomal protein (Tv40Srp) gene or RNA.In some embodiments, the method comprises contacting nucleic acids fromthe sample with a second primer pair for detecting an endogenouscontrol. In some embodiments, the method comprises contacting nucleicacids from the sample with a third primer pair for detecting anexogenous control.

In some embodiments, the first primer pair comprises a first primer anda second primer, wherein the first primer comprises a sequence that isat least 90%, at least 95%, or 100% identical to at least 15, at least16, at least 17, at least 18, at least 19, at least 20, at least 21, atleast 22, at least 23, at least 24, or at least 25 contiguousnucleotides of SEQ ID NO: 4, and wherein the second primer comprises asequence that is at least 90%, at least 95%, or 100% complementary to atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, at least 21, at least 22, at least 23, at least 24, or at least 25contiguous nucleotides of SEQ ID NO: 4. In some embodiments, the firstprimer and the second primer each independently comprises 0, 1, or 2mismatches compared to SEQ ID NO: 4 or its complement. In someembodiments, the first primer pair comprises a first primer consistingof 15 to 30 nucleotides and a second primer consisting of 15 to 30nucleotides. In some embodiments, the first primer pair comprises afirst primer of SEQ ID NO: 1 and a second primer of SEQ ID NO: 2. Insome embodiments, the first primer pair produces an amplicon that is 50to 500 nucleotides long, 50 to 400 nucleotides long, 50 to 300nucleotides long, 50 to 200 nucleotides long, 50 to 150 nucleotideslong, 100 to 300 nucleotides long, 100 to 200 nucleotides long, or 100to 150 nucleotides long.

In some embodiments, the method comprises forming the Tv40Srp amplicon.In some embodiments, the method comprises contacting the Tv40Srpamplicon with a first probe capable of selectively hybridizing with theTv40Srp amplicon. In some embodiments, the first probe comprises adetectable label. In some embodiments, the first probe comprises afluorescent dye and a quencher molecule. In some embodiments, the firstprobe comprises a sequence that is at least 90%, at least 95%, or 100%identical or complementary to at least 15, at least 16, at least 17, atleast 18, at least 19, at least 20, at least 21, at least 22, at least23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 4or SEQ ID NO: 5. In some embodiments, the first probe comprises 0, 1, or2 mismatches compared to SEQ ID NO: 4 or its complement or compared toSEQ ID NO: 5 or its complement. In some embodiments, the first probeconsists of 15 to 30 nucleotides. In some embodiments, the first probehas the sequence of SEQ ID NO: 3.

In some embodiments, the method comprises forming an endogenous controlamplicon and/or an exogenous control amplicon. In some embodiments, themethod comprises contacting the endogenous control amplicon with asecond probe capable of selectively hybridizing with the engoenouscontrol amplicon and/or contacting the exogenous control amplicon with athird probe capable of selectively hybridizing with the exoenous controlamplicon. In some embodiments, the second probe and the third probe eachcomprise a detectable label, wherein the detectable labels may be thesame or different. In some embodiments, the detectable labels of thesecond and third probes are detectably different from the detectablelabel of the first probe. In some embodiments, the method comprisesdetecting the Tv40Srp gene or RNA, an endogenous control, and anexogenous control in a single multiplex reaction.

In some embodiments, the sample is selected from a urine sample, anendocervical swab sample, a vaginal swab sample, and a urethral swabsample.

In some embodiments, compositions comprising a first primer pair fordetecting a Trichomonas vaginalis 40S ribosomal protein (Tv40Srp) geneor RNA are provided. In some embodiments, the composition comprises asecond primer pair for detecting an endogenous control. In someembodiments, the endogenous control is a sample adequacy control. Insome embodiments, the endogenous control is selected from HMBS, GAPDH,beta actin, and beta globin. In some embodiments, the compositioncomprises a third primer pair for detecting an exogenous control. Insome embodiments, the exogenous control is a sample processing control.In some embodiments, the exogenous control is a bacterial gene.

In some embodiments, the first primer pair comprises a first primer anda second primer, wherein the first primer comprises a sequence that isat least 90%, at least 95%, or 100% identical to at least 15, at least16, at least 17, at least 18, at least 19, at least 20, at least 21, atleast 22, at least 23, at least 24, or at least 25 contiguousnucleotides of SEQ ID NO: 4, and wherein the second primer comprises asequence that is at least 90%, at least 95%, or 100% complementary to atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, at least 21, at least 22, at least 23, at least 24, or at least 25contiguous nucleotides of SEQ ID NO: 4. In some embodiments, the firstprimer and the second primer each independently comprises 0, 1, or 2mismatches compared to SEQ ID NO: 4 or its complement. In someembodiments, the first primer pair comprises a first primer consistingof 15 to 30 nucleotides and a second primer consisting of 15 to 30nucleotides. In some embodiments, the first primer pair comprises afirst primer of SEQ ID NO: 1 and a second primer of SEQ ID NO: 2.

In some embodiments, the composition comprises a first probe capable ofselectively hybridizing to a Tv40Srp amplicon produced by the firstprimer pair. In some embodiments, the first probe comprises a detectablelabel. In some embodiments, the first probe comprises a fluorescent dyeand a quencher molecule. In some embodiments, the first probe comprisesa sequence that is at least 90%, at least 95%, or 100% identical orcomplementary to at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, or at least 25 contiguous nucleotides of SEQ ID NO: 4 or SEQ ID NO:5. In some embodiments, the first probe comprises 0, 1, or 2 mismatchescompared to SEQ ID NO: 4 or its complement or compared to SEQ ID NO: 5or its complement. In some embodiments, the first probe consists of 15to 30 nucleotides. In some embodiments, the first probe has the sequenceof SEQ ID NO: 3. In some embodiments, the Tv40Srp amplicon has thesequence of SEQ ID NO: 5.

In some embodiments, the composition comprises a second probe capable ofselectively hybridizing to an endogenous control amplicon produced bythe second primer pair. In some embodiments, the endogenous control is asample adequacy control. In some embodiments, the endogenous control isselected from HMBS, GAPDH, beta actin, and beta globin. In someembodiments, the composition comprises a third probe capable ofselectively hybridizing to an exogenous control amplicon produced by thethird primer pair. In some embodiments, the exogenous control is asample processing control. In some embodiments, the exogenous controlcomprises a DNA sequence that is not expected to be present in thesample. In some embodiments, the exogenous control is a bacterial DNA.

In some embodiments, the composition is a lyophilized composition. Insome embodiments, the composition is in solution. In some embodiments,the composition comprises nucleic acids from a sample from a subjectbeing tested for the presence of absence of Trichomonas vaginalis.

In some embodiments, kits are provided comprising a first primer pairfor detecting a Trichomonas vaginalis 40S ribosomal protein (Tv40Srp)gene or RNA. In some embodiments, the kit comprises a second primer pairfor detecting an endogenous control, wherein the primer pair fordetecting Tv40Srp and the second primer pair are in the same ordifferent compositions in the kit. In some embodiments, the endogenouscontrol is a sample adequacy control. In some embodiments, theendogenous control is selected from HMBS, GAPDH, beta actin, and betaglobin. In some embodiments, the kit comprises a third primer pair fordetecting an exogenous control, wherein the third primer pair is in thesame or different composition from the primer pair for detecting Tv40Srpand the second primer pair. In some embodiments, the exogenous controlis a sample processing control. In some embodiments, the exogenouscontrol comprises a DNA sequence that is not expected to be present inthe sample. In some embodiments, the exogenous control is a bacterialgene.

In some embodiments, the first primer pair comprises a first primer anda second primer, wherein the first primer comprises a sequence that isat least 90%, at least 95%, or 100% identical to at least 15, at least16, at least 17, at least 18, at least 19, at least 20, at least 21, atleast 22, at least 23, at least 24, or at least 25 contiguousnucleotides of SEQ ID NO: 4, and wherein the second primer comprises asequence that is at least 90%, at least 95%, or 100% complementary to atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, at least 21, at least 22, at least 23, at least 24, or at least 25contiguous nucleotides of SEQ ID NO: 4. In some embodiments, the firstprimer and the second primer each independently comprises 0, 1, or 2mismatches compared to SEQ ID NO: 4 or its complement. In someembodiments, the first primer pair comprises a first primer consistingof 15 to 30 nucleotides and a second primer consisting of 15 to 30nucleotides. In some embodiments, the first primer pair comprises afirst primer of SEQ ID NO: 1 and a second primer of SEQ ID NO: 2.

In some embodiments, the kit comprises a first probe capable ofselectively hybridizing to a Tv40Srp amplicon produced by the firstprimer pair, wherein the first probe is in the same or differentcomposition from one or more of the primer pairs. In some embodiments,the first probe comprises a detectable label. In some embodiments, thefirst probe comprises a fluorescent dye and a quencher molecule. In someembodiments, the first probe comprises a sequence that is at least 90%,at least 95%, or 100% identical or complementary to at least 15, atleast 16, at least 17, at least 18, at least 19, at least 20, at least21, at least 22, at least 23, at least 24, or at least 25 contiguousnucleotides of SEQ ID NO: 4 or SEQ ID NO: 5. In some embodiments, thefirst probe comprises 0, 1, or 2 mismatches compared to SEQ ID NO: 4 orits complement or compared to SEQ ID NO: 5 or its complement. In someembodiments, the first probe consists of 15 to 30 nucleotides. In someembodiments, the first probe has the sequence of SEQ ID NO: 3. In someembodiments, the Tv40Srp amplicon has the sequence of SEQ ID NO: 5.

In some embodiments, the kit comprises a second probe capable ofselectively hybridizing to an endogenous control amplicon produced bythe second primer pair, wherein the second probe is in the same ordifferent composition from one or more of the primer pairs. In someembodiments, the kit comprises a third probe capable of selectivelyhybridizing to an exogenous control amplicon produced by the thirdprimer pair, wherein the third probe is in the same or differentcomposition from one or more of the primer pairs.

In some embodiments, the kit comprises dNTPs and/or a thermostablepolymerase. In some embodiments, the kit comprises one or morelyophilized compositions.

In some embodiments, a primer is provided, wherein the primer consistsof the sequence of SEQ ID NO: 1, wherein the primer comprises at leastone modified nucleotide. In some embodiments, a primer is provided,wherein the primer consists of the sequence of SEQ ID NO: 2, wherein theprimer comprises at least one modified nucleotide. In some embodiments,a probe is provided, wherein the probe consists of the sequence of SEQID NO: 3, wherein the probe comprises at least one modified nucleotideand/or a detectable label. In some embodiments, the probe comprises afluorescent dye and a quencher molecule. In some embodiments, the probeis a fluorescence resonance energy transfer (FRET) probe. In someembodiments, the probe comprises at least one modified nucleotide.

In some embodiments, a composition is provided, wherein the compositioncomprises a first primer consisting of the sequence of SEQ ID NO: 2 anda second primer consisting of the sequence of SEQ ID NO: 3, wherein thefirst primer and the second primer each comprises at least one modifiednucleotide. In some embodiments, the composition comprises a probeconsisting of the sequence of SEQ ID NO: 3, wherein the probe comprisesat least one modified nucleotide and/or a detectable label. In someembodiments, the probe comprises a fluorescent dye and a quenchermolecule. In some embodiments, the probe is a fluorescence resonanceenergy transfer (FRET) probe. In some embodiments, the probe comprisesat least one modified nucleotide. In some embodiments, the compositionis a lyophilized composition. In some embodiments, the composition is insolution. In some embodiments, the composition comprises nucleic acidsof a sample from a subject.

Further embodiments and details of the inventions are described below.

4. DETAILED DESCRIPTION 4.1. Definitions

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

As used herein, the terms “detect”, “detecting” or “detection” maydescribe either the general act of discovering or discerning or thespecific observation of a detectably labeled composition.

As used herein, the term “detectably different” refers to a set oflabels (such as dyes) that can be detected and distinguishedsimultaneously.

As used herein, the terms “patient” and “subject” are usedinterchangeably to refer to a human. In some embodiments, the methodsdescribed herein may be used on samples from non-human animals.

“Trichomonas vaginalis” refers to the protozoan responsible fortrichomoniasis, a common sexually transmitted infection that can infectboth men and women. Trichomoniasis may be symptomatic or asymptomatic.Symptoms of trichomoniasis include, but are not limited to, vaginitis,urethritis, and cervicitis. Symptoms in females include, but are notlimited to, itching, burning, redness, or soreness of the genitals,unusual odor, discomfort with urination, or a thin clear, white, yellow,or green discharge. Symptoms in males include, but are not limited to,itching or burning inside the penis, burning after ejaculation orurination, or penile discharge.

As used herein, the terms “oligonucleotide,” “polynucleotide,” “nucleicacid molecule,” and the like, refer to nucleic acid-containingmolecules, including but not limited to, DNA or RNA. The termencompasses sequences that include any of the known base analogs of DNAand RNA including, but not limited to, 4-acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

As used herein, the term “oligonucleotide,” refers to a single-strandedpolynucleotide having fewer than 500 nucleotides. In some embodiments,an oligonucleotide is 8 to 200, 8 to 100, 12 to 200, 12 to 100, 12 to75, or 12 to 50 nucleotides long. Oligonucleotides may be referred to bytheir length, for example, a 24 residue oligonucleotide may be referredto as a “24-mer.”

As used herein, the term “complementary” to a target gene (or targetregion thereof), and the percentage of “complementarity” of the probesequence to the target gene sequence is the percentage “identity” to thesequence of target gene or to the reverse complement of the sequence ofthe target gene. In determining the degree of “complementarity” betweenprobes used in the compositions described herein (or regions thereof)and a target gene, such as those disclosed herein, the degree of“complementarity” is expressed as the percentage identity between thesequence of the probe (or region thereof) and sequence of the targetgene or the reverse complement of the sequence of the target gene thatbest aligns therewith. The percentage is calculated by counting thenumber of aligned bases that are identical as between the 2 sequences,dividing by the total number of contiguous nucleotides in the probe, andmultiplying by 100. When the term “complementary” is used, the subjectoligonucleotide is at least 90% complementary to the target molecule,unless indicated otherwise. In some embodiments, the subjectoligonucleotide is at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% complementary to the target molecule.

A “primer” or “probe” as used herein, refers to an oligonucleotide thatcomprises a region that is complementary to a sequence of at least 8contiguous nucleotides of a target nucleic acid molecule, such as DNA(e.g., a target gene) or an mRNA (or a DNA reverse-transcribed from anmRNA). In some embodiments, a primer or probe comprises a region that iscomplementary to a sequence of at least 9, at least 10, at least 11, atleast 12, at least 13, at least 14, at least 15, at least 16, at least17, at least 18, at least 19, at least 20, at least 21, at least 22, atleast 23, at least 24, at least 25, at least 26, at least 27, at least28, at least 29, or at least 30 contiguous nucleotides of a targetmolecule. When a primer or probe comprises a region that is“complementary to at least x contiguous nucleotides of a targetmolecule,” the primer or probe is at least 95% complementary to at leastx contiguous nucleotides of the target molecule. In some embodiments,the primer or probe is at least 96%, at least 97%, at least 98%, atleast 99%, or 100% complementary to the target molecule.

The term “nucleic acid amplification,” encompasses any means by which atleast a part of at least one target nucleic acid is reproduced,typically in a template-dependent manner, including without limitation,a broad range of techniques for amplifying nucleic acid sequences,either linearly or exponentially. Exemplary means for performing anamplifying step include polymerase chain reaction (PCR), ligase chainreaction (LCR), ligase detection reaction (LDR), multiplexligation-dependent probe amplification (MLPA), ligation followed byQ-replicase amplification, primer extension, strand displacementamplification (SDA), hyperbranched strand displacement amplification,multiple displacement amplification (MDA), nucleic acid strand-basedamplification (NASBA), two-step multiplexed amplifications, rollingcircle amplification (RCA), and the like, including multiplex versionsand combinations thereof, for example but not limited to, OLA/PCR,PCR/OLA, LDR/PCR, PCR/PCR/LDR, PCR/LDR, LCR/PCR, PCR/LCR (also known ascombined chain reaction—CCR), digital amplification, and the like.Descriptions of such techniques can be found in, among other sources,Ausbel et al.; PCR Primer: A Laboratory Manual, Diffenbach, Ed., ColdSpring Harbor Press (1995); The Electronic Protocol Book, ChangBioscience (2002); Msuih et al., J. Clin. Micro. 34:501-07 (1996); TheNucleic Acid Protocols Handbook, R. Rapley, ed., Humana Press, Totowa,N.J. (2002); Abramson et al., Curr Opin Biotechnol. 1993 February;4(1):41-7, U.S. Pat. Nos. 6,027,998; 6,605,451, Barany et al., PCTPublication No. WO 97/31256; Wenz et al., PCT Publication No. WO01/92579; Day et al., Genomics, 29(1): 152-162 (1995), Ehrlich et al.,Science 252:1643-50 (1991); Innis et al., PCR Protocols: A Guide toMethods and Applications, Academic Press (1990); Favis et al., NatureBiotechnology 18:561-64 (2000); and Rabenau et al., Infection 28:97-102(2000); Belgrader, Barany, and Lubin, Development of a MultiplexLigation Detection Reaction DNA Typing Assay, Sixth InternationalSymposium on Human Identification, 1995 (available on the world wide webat: promega.com/geneticidproc/ussymp6proc/blegrad.html); LCR KitInstruction Manual, Cat. #200520, Rev. #050002, Stratagene, 2002;Barany, Proc. Natl. Acad. Sci. USA 88:188-93 (1991); Bi and Sambrook,Nucl. Acids Res. 25:2924-2951 (1997); Zirvi et al., Nucl. Acid Res.27:e40i-viii (1999); Dean et al., Proc Natl Acad Sci USA 99:5261-66(2002); Barany and Gelfand, Gene 109:1-11 (1991); Walker et al., Nucl.Acid Res. 20:1691-96 (1992); Polstra et al., BMC Inf. Dis. 2:18-(2002);Lage et al., Genome Res. 2003 February; 13(2):294-307, and Landegren etal., Science 241:1077-80 (1988), Demidov, V., Expert Rev Mol Diagn. 2002November; 2(6):542-8., Cook et al., J Microbiol Methods. 2003 May;53(2):165-74, Schweitzer et al., Curr Opin Biotechnol. 2001 February;12(1):21-7, U.S. Pat. Nos. 5,830,711, 6,027,889, 5,686,243, PCTPublication No. WO0056927A3, and PCT Publication No. WO9803673A1.

In some embodiments, amplification comprises at least one cycle of thesequential procedures of: annealing at least one primer withcomplementary or substantially complementary sequences in at least onetarget nucleic acid; synthesizing at least one strand of nucleotides ina template-dependent manner using a polymerase; and denaturing thenewly-formed nucleic acid duplex to separate the strands. The cycle mayor may not be repeated. Amplification can comprise thermocycling or canbe performed isothermally.

Unless otherwise indicated, the term “hybridize” is used herein refer to“specific hybridization” which is the binding, duplexing, or hybridizingof a nucleic acid molecule preferentially to a particular nucleotidesequence, in some embodiments, under stringent conditions. The term“stringent conditions” refers to conditions under which a probe willhybridize preferentially to its target sequence, and to a lesser extentto, or not at all to, other sequences. A “stringent hybridization” and“stringent hybridization wash conditions” in the context of nucleic acidhybridization (e.g., as in array, Southern, or Northern hybridization)are sequence-dependent and are different under different environmentalparameters. An extensive guide to the hybridization of nucleic acids isfound in, e.g., Tijssen (1993) Laboratory Techniques in Biochemistry andMolecular Biology—Hybridization with Nucleic Acid Probes part I, Ch. 2,“Overview of principles of hybridization and the strategy of nucleicacid probe assays,” Elsevier, N.Y. (“Tijssen”). Generally, highlystringent hybridization and wash conditions for filter hybridizationsare selected to be about 5° C. lower than the thermal melting point(T_(m)) for the specific sequence at a defined ionic strength and pH.The T_(m) is the temperature (under defined ionic strength and pH) atwhich 50% of the target sequence hybridizes to a perfectly matchedprobe. Very stringent conditions are selected to be equal to the T_(m)for a particular probe. Dependency of hybridization stringency on buffercomposition, temperature, and probe length are well known to those ofskill in the art (see, e.g., Sambrook and Russell (2001) MolecularCloning: A Laboratory Manual (3rd ed.) Vol. 1-3, Cold Spring HarborLaboratory, Cold Spring Harbor Press, N.Y.).

A “sample,” as used herein, includes urine samples (including samplesderived from urine samples), endocervical swabs, and patient-collectedvaginal swabs, and other types of human samples. In some embodiments, aurine sample is a “first catch” urine sample, which is a sample taken asa subject first begins to urinate. As used herein, urine samplesinclude, but are not limited to, whole urine, a sample comprising cellsfrom a urine sample, a sample comprising the cell pellet isolated bycentrifugation of a urine sample, a sample comprising cells isolated byfiltration of a urine sample, and the like. In some embodiments, a urinesample comprises a buffer, such as a preservative. In some embodiments,a sample is a human sample other than a urine sample, such as anendocervical swab or a vaginal swab, including a patient-collectedvaginal swab, and a urethral swab. In some embodiments, a swab samplecomprises a buffer, such as a preservative.

An “endogenous control,” as used herein refers to a moiety that isnaturally present in the sample to be used for detection. In someembodiments, an endogenous control is a “sample adequacy control” (SAC),which may be used to determine whether there was sufficient sample usedin the assay, or whether the sample comprised sufficient biologicalmaterial, such as cells. In some embodiments, an SAC is a single copyhuman gene. In some embodiments, an endogenous control, such as an SAC,is selected that can be detected in the same manner as the target geneis detected and, in some embodiments, simultaneously with the targetgene.

An “exogenous control,” as used herein, refers to a moiety that is addedto a sample or to an assay, such as a “sample processing control” (SPC).In some embodiments, an exogenous control is included with the assayreagents. An exogenous control is typically selected that is notexpected to be present in the sample to be used for detection, or ispresent at very low levels in the sample such that the amount of themoiety naturally present in the sample is either undetectable or isdetectable at a much lower level than the amount added to the sample asan exogenous control. In some embodiments, an exogenous controlcomprises a nucleotide sequence that is not expected to be present inthe sample type used for detection of the target gene. In someembodiments, an exogenous control comprises a nucleotide sequence thatis not known to be present in the species from whom the sample is taken.In some embodiments, an exogenous control comprises a nucleotidesequence from a different species than the subject from whom the samplewas taken. In some embodiments, an exogenous control comprises anucleotide sequence that is not known to be present in any species. Insome embodiments, an exogenous control is selected that can be detectedin the same manner as the target gene is detected and, in someembodiments, simultaneously with the target gene. In some embodiments,an exogenous control is a bacterial DNA. In some embodiments, thebacterium is a species not expected to be found in the sample type beingtested.

In the sequences herein, “U” and “T” are used interchangeably, such thatboth letters indicate a uracil or thymine at that position. One skilledin the art will understand from the context and/or intended use whethera uracil or thymine is intended and/or should be used at that positionin the sequence. For example, one skilled in the art would understandthat native RNA molecules typically include uracil, while native DNAmolecules typically include thymine. Thus, where an RNA sequenceincludes “T”, one skilled in the art would understand that that positionin the native RNA is likely a uracil.

In the present disclosure, “a sequence selected from” encompasses both“one sequence selected from” and “one or more sequences selected from.”Thus, when “a sequence selected from” is used, it is to be understoodthat one, or more than one, of the listed sequences may be chosen.

4.2. Detecting Trichomonas vaginalis

The present inventors have developed an assay for detecting Trichomonasvaginalis (TV). In some embodiments, the assay comprises detecting theTV 40S ribosomal protein (Tv40Srp) gene. In some embodiments, the assaycomprises detecting RNA transcribed from the TV 40S ribosomal protein(Tv40Srp) gene. The present assay relies on the polymerase chainreaction (PCR), and can be carried out in a substantially automatedmanner using a commercially available nucleic acid amplification system.Exemplary nonlimiting nucleic acid amplification systems that can beused to carry out the methods of the invention include the GeneXpert®system, a GeneXpert® Infinity system, and a Smartcycler System (Cepheid,Sunnyvale, Calif.). The present assay can be completed in under 3 hours,and in some embodiments, under 2 hours, using an automated system, forexample, the GeneXpert® system.

4.2.1. General Methods

Compositions and methods for detecting Trichomonas vaginalis (TV) areprovided. In some embodiments, the method comprises detecting the TV 40Sribosomal protein (Tv40Srp) gene.

In some embodiments, a method of detecting Trichomonas vaginalis (TV) ina subject comprises detecting the presence of the TV 40S ribosomalprotein (Tv40Srp) gene in a sample from the subject. In someembodiments, the sample is selected from a urine sample, an endocervicalswab, and a vaginal swab. In some embodiments, the urine sample is afirst catch urine sample.

In some embodiments, a method of detecting TV further comprisesdetecting at least one endogenous control, such as a sample adequacycontrol (SAC). In some embodiments, a method of detecting TV furthercomprises detecting at least one exogenous control, such as a sampleprocessing control (SPC). In some embodiments, a method of detecting TVfurther comprises detecting at least one endogenous control and at leastone exogenous control.

In some embodiments, a method of detecting TV comprises detecting the TV40S ribosomal protein (Tv40Srp) gene in a sample. In some embodiments, amethod of detecting TV further comprises detecting a sample adequacycontrol (SAC), such as a single copy human gene. In some embodiments, amethod of detecting TV further comprises detecting a sample processingcontrol (SPC), such as an exogenously added bacterial DNA. In someembodiments, a method of detecting TV further comprises detecting an SACand an SPC.

In the present disclosure, the term “target gene” is used forconvenience to refer to the TV 40S ribosomal protein (Tv40Srp) gene, andalso to exogenous and/or endogenous controls. Thus, it is to beunderstood that when a discussion is presented in terms of a targetgene, that discussion is specifically intended to encompass the TV 40Sribosomal protein (Tv40Srp) gene, the endogenous control(s) (e.g., SAC),and the exogenous control(s) (e.g., SPC).

In some embodiments, the presence of the TV 40S ribosomal protein(Tv40Srp) gene is detected in a urine sample. In some embodiments, thetarget gene is detected in a urine sample to which a buffer (such as apreservative) has been added. In some embodiments, the buffer is addedto a urine sample at a ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8,1:9, or 1:10 buffer:urine. In some embodiments, the presence of the TV40S ribosomal protein (Tv40Srp) gene is detected in an endocervical swabsample or a vaginal swab sample. In some embodiments, the vaginal swabis a patient-collected vaginal swab. In some embodiments, the targetgene is detected in an endocervical swab sample or a vaginal swab samplethat has been placed in a buffer (such as a preservative). In someembodiments, a swab is placed in 1 mL, 2 mL, 2.5 mL of buffer.

In some embodiments, detection of the TV 40S ribosomal protein (Tv40Srp)gene in a sample from a subject indicates the presence of Trichomonasvaginalis in the subject. In some embodiments, the detecting is donequantitatively. In other embodiments, the detecting is donequalitatively. In some embodiments, detecting a target gene comprisesforming a complex comprising a polynucleotide and a nucleic acidselected from a target gene, a DNA amplicon of a target gene, and acomplement of a target gene. In some embodiments, detecting a targetgene comprises PCR. In some embodiments, detecting a target genecomprises quantitative PCR or real-time PCR. In some embodiments, asample adequacy control (SAC) and/or a sample processing control (SPC)is detected in the same assay as the target gene. In some embodiments,if the TV 40S ribosomal protein (Tv40Srp) gene is detected, TV isconsidered to be detected even if the SPC and/or SAC are not detected inthe assay. In some embodiments, if the TV 40S ribosomal protein(Tv40Srp) gene is not detected, TV is considered to be not detected onlyif the SPC and SAC are also detected in the assay.

In some embodiments, the presence of the TV 40S ribosomal protein(Tv40Srp) gene can be measured in samples collected at one or more timesfrom a subject to monitor treatment for TV infection in the subject.Treatments include, but are not limited to, a single dose or multipledoses of metronidazole or tinidazole. In some embodiments, a subjectwith a history of TV infection is monitored for recurrence of TV bydetecting the presence or absence of the TV 40S ribosomal protein(Tv40Srp) gene at regular or semi-regular intervals. In some suchembodiments, the patient is monitored by detecting the presence orabsence of the TV 40S ribosomal protein (Tv40Srp) gene at least once permonth, at least once every two months, at least once every three months,at least once every four months, at least once every five months, atleast once every six months, at least once every nine months, at leastonce per year, or at least once every two years.

In some embodiments, the present assay may be used as part of routineand/or preventative healthcare for a subject. That is, in someembodiments, the present assay may be used to test an individual for TVinfection whether or not the individual has exhibited symptoms of TVinfection or has a history of TV infection. In some embodiments, thepresent assay is used to detect TV infection in subjects who arepregnant and/or who are attempting to become pregnant. In someinstances, pregnant women with TV are more likely to experience pre-termdelivery and/or have babies with low birth weight (less than 5.5pounds).

In some embodiments, a sample to be tested is a urine sample (such as afirst catch urine sample), or is derived from a urine sample. In someembodiments, a buffer (such as a preservative) is added to the urinesample. In some embodiments, the buffer is added to the urine samplewithin 1 hour, within 2 hours, within 3 hours, within 4 hours, within 5hours, within 6 hours, within 7 hours, or within 8 hours of samplecollection.

In some embodiments, a sample to be tested is an endocervical swabsample or a vaginal swab sample. In some embodiments, the swab is placedin a buffer. In some embodiments, the swab is immediately placed in thebuffer. In some embodiments, the swab is placed in the buffer within 1hour, within 2 hours, within 3 hours, within 4 hours, within 5 hours,within 6 hours, within 7 hours, or within 8 hours of sample collection.

In some embodiments, less than 5 ml, less than 4 ml, less than 3 ml,less than 2 ml, less than 1 ml, or less than 0.75 ml of urine are usedin the present methods. In some embodiments, 0.1 ml to 1 ml of urine isused in the present methods.

In some embodiments, the sample to be tested is another bodily fluid,such as blood, sputum, mucus, saliva, vaginal or penile discharge,semen, etc.

The clinical sample to be tested is, in some embodiments, fresh (i.e.,never frozen). In other embodiments, the sample is a frozen specimen. Insome embodiments, the sample is a tissue sample, such as aformalin-fixed paraffin embedded sample. In some embodiments, the sampleis a liquid cytology sample.

In some embodiments, the sample to be tested is obtained from anindividual who has one or more symptoms of TV infection. Nonlimitingexemplary symptoms of TV infection include vaginitis, urethritis, andcervicitis; in females: itching, burning, redness, or soreness of thegenitals, unusual odor, discomfort with urination, and a thin clear,white, yellow, or green discharge; and in males: itching or burninginside the penis, burning after ejaculation or urination, and peniledischarge. In some embodiments, the sample to be tested is obtained froman individual who has previously been diagnosed with TV infection. Insome such embodiments, the individual is monitored for recurrence of TVinfection.

In some embodiments, methods described herein can be used for routinescreening of healthy individuals with no risk factors. In someembodiments, methods described herein are used to screen asymptomaticindividuals, for example, during routine or preventative care. In someembodiments, methods described herein are used to screen women who arepregnant or who are attempting to become pregnant.

In some embodiments, the methods described herein can be used to assessthe effectiveness of a treatment for TV infection in a patient.

In some embodiments, use of the the TV 40S ribosomal protein (Tv40Srp)gene for detecting TV infection is provided. In some embodiments, use ofthe the TV 40S ribosomal protein (Tv40Srp) gene for monitoringrecurrence of TV infection is provided.

In any of the embodiments described herein, the TV 40S ribosomal protein(Tv40Srp) gene may be detected in the same assay reaction as a sampleprocessing control (SPC) and/or sample adequacy control (SAC).

In some embodiments, a method of facilitating detection of TV infectionin a subject is provided. Such methods comprise detecting the presenceor absence of the TV 40S ribosomal protein (Tv40Srp) gene in a samplefrom the subject. In some embodiments, information concerning thepresence or absence of the TV 40S ribosomal protein (Tv40Srp) gene inthe sample from the subject is communicated to a medical practitioner. A“medical practitioner,” as used herein, refers to an individual orentity that diagnoses and/or treats patients, such as a hospital, aclinic, a physician's office, a physician, a nurse, or an agent of anyof the aforementioned entities and individuals. In some embodiments,detecting the presence or absence of TV 40S ribosomal protein (Tv40Srp)gene is carried out at a laboratory that has received the subject'ssample from the medical practitioner or agent of the medicalpractitioner. The laboratory carries out the detection by any method,including those described herein, and then communicates the results tothe medical practitioner. A result is “communicated,” as used herein,when it is provided by any means to the medical practitioner. In someembodiments, such communication may be oral or written, may be bytelephone, in person, by e-mail, by mail or other courier, or may bemade by directly depositing the information into, e.g., a databaseaccessible by the medical practitioner, including databases notcontrolled by the medical practitioner. In some embodiments, theinformation is maintained in electronic form. In some embodiments, theinformation can be stored in a memory or other computer readable medium,such as RAM, ROM, EEPROM, flash memory, computer chips, digital videodiscs (DVD), compact discs (CDs), hard disk drives (HDD), magnetic tape,etc.

In some embodiments, methods of detecting TV are provided. In someembodiments, methods of diagnosing TV infection are provided. In someembodiments, the method comprises obtaining a sample from a subject andproviding the sample to a laboratory for detection of the TV 40Sribosomal protein (Tv40Srp) gene in the sample. In some embodiments, themethod further comprises receiving a communication from the laboratorythat indicates the presence or absence of the TV 40S ribosomal protein(Tv40Srp) gene in the sample. A “laboratory,” as used herein, is anyfacility that detects the target gene in a sample by any method,including the methods described herein, and communicates the result to amedical practitioner. In some embodiments, a laboratory is under thecontrol of a medical practitioner. In some embodiments, a laboratory isnot under the control of the medical practitioner.

When a laboratory communicates the result of detecting the presence orabsence of the TV 40S ribosomal protein (Tv40Srp) gene to a medicalpractitioner, in some embodiments, the laboratory indicates whether ornot the TV 40S ribosomal protein (Tv40Srp) gene was detected in thesample. In some embodiments, the laboratory indicates whether the samplecomprises Trichomonas vaginalis (TV), by indicating, for example, “TVpositive” or “TV negative” or “TV present” or “TV absent,” and the like.

As used herein, when a method relates to detecting TV, determining thepresence of TV, monitoring for TV, and/or diagnosing TV infection, themethod includes activities in which the steps of the method are carriedout, but the result is negative for the presence of TV. That is,detecting, determining, monitoring, and diagnosing TV or TV infectioninclude instances of carrying out the methods that result in eitherpositive or negative results.

In some embodiments, at least one endogenous control (e.g., an SAC)and/or at least one exogenous control (e.g., an SPC) are detectedsimultaneously with the TV 40S ribosomal protein (Tv40Srp) gene in asingle reaction.

4.2.2. Exemplary Controls

In some embodiments, an assay described herein comprises detecting theTV 40S ribosomal protein (Tv40Srp) gene and at least one endogenouscontrol. In some embodiments, the endogenous control is a sampleadequacy control (SAC). In some such embodiments, if the TV 40Sribosomal protein (Tv40Srp) gene is not detected in a sample, and theSAC is also not detected in the sample, the assay result is considered“invalid” because the sample may have been insufficient. While notintending to be bound by any particular theory, an insufficient samplemay be too dilute, contain too little cellular material, contain anassay inhibitor, etc. In some embodiments, the failure to detect an SACmay indicate that the assay reaction failed. In some embodiments, anendogenous control (such as an SAC) is a single-copy human gene.Nonlimiting exemplary SACs include human hydroxymethyl-bilane synthase(HMBS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta actin,beta2-microglobin, cyclooxygenase 1, hypoxanthinephosphoribosyl-transferase, porphobilinogen deaminase, and thetransferrin receptor.

In some embodiments, an assay described herein comprises detecting theTV 40S ribosomal protein (Tv40Srp) gene and at least one exogenouscontrol. In some embodiments, the exogenous control is a sampleprocessing control (SPC). In some such embodiments, if the TV 40Sribosomal protein (Tv40Srp) gene is not detected in a sample, and theSPC is also not detected in the sample, the assay result is considered“invalid” because there may have been an error in sample processing,including but not limited to, failure of the assay. Nonlimitingexemplary errors in sample processing include, inadequate sampleprocessing, the presence of an assay inhibitor, compromised reagents,etc. In some embodiments, an exogenous control (such as an SPC) is addedto a sample. In some embodiments, an exogenous control (such as an SPC)is added during performance of an assay, such as with one or morebuffers or reagents. In some embodiments, when a GeneXpert® system is tobe used, the SPC is included in the GeneXpert® cartridge. In someembodiments, an exogenous control (such as an SPC) is a DNA sequencethat is not expected to be present in the sample being assayed.Nonlimiting exemplary SPCs include bacterial genes not expected to bepresent in the sample being assayed.

In some embodiments, an endogenous control and/or an exogenous controlis detected contemporaneously, such as in the same assay, as detectionof the TV 40S ribosomal protein (Tv40Srp) gene in a sample. In someembodiments, an assay comprises reagents for detecting the TV 40Sribosomal protein (Tv40Srp) gene, an exogenous control, and anendogenous control simultaneously in the same assay reaction. In somesuch embodiments, for example, an assay reaction comprises a primer setfor amplifying the TV 40S ribosomal protein (Tv40Srp) gene, a primer setfor amplifying an endogenous control, and a primer set for amplifying anexogenous control, and labeled probes for detecting the amplificationproducts (such as, for example, TaqMan® probes).

4.2.3. Exemplary Sample Preparation

4.2.3.1. Exemplary Buffers

In some embodiments, a buffer is added to a urine sample. In someembodiments, the buffer is added within one hour, two hours, threehours, or six hours of the time the urine sample was collected (e.g.,voided). In some embodiments, a buffer is added to the urine samplewithin one hour, two hours, three hours, or six hours before the sampleis analyzed by the methods described herein.

In some embodiments, a swab sample is placed in a buffer. In someembodiments, the swab sample is placed in the buffer within one hour,two hours, three hours, or six hours of the time the swab sample wascollected. In some embodiments, the swab sample is placed in a bufferwithin one hour, two hours, three hours, or six hours before the sampleis analyzed by the methods described herein.

Non-limiting exemplary commercial buffers include PreservCyt (Hologic,Bedford, Mass.), SurePath (BD, Franklin Lakes, N.J.), and CyMol (CopanDiagnostics, Murrietta, Calif.).

4.2.3.2. Exemplary DNA Preparation

Sample DNA can be prepared by any appropriate method. In someembodiments, target DNA is prepared by contacting a sample with a lysisbuffer and binding DNA to a DNA binding substrate, such as a glass orsilica substrate. The binding substrate may have any suitable form, suchas a particulate, porous solid, or membrane form. For example, thesupport may comprise hydroxycellulose, glass fiber, cellulose,nitrocellulose, zirconium hydroxide, titanium (IV) oxide, silicondioxide, zirconium silicate, or silica particles (e.g., see U.S. Pat.No. 5,234,809). Many such DNA binding substrates are known in the art.

In some embodiments, DNA is detected in a lysate without first isolatingor separating the DNA. In some some embodiments, the sample is subjectto a lysis step to release the DNA. Non-limiting exemplary lysis methodsinclude sonication (for example, for 2-15 seconds, 8-18 μm at 36 kHz);chemical lysis, for example, using a detergent; and various commerciallyavailable lysis reagents. In some embodiments, DNA is detected aremeasured in a sample in which DNA has been isolated or separated from atleast some other cellular components.

When the methods discussed herein indicate that a target gene isdetected, such detection may be carried out on a complement of a targetgene instead of, or in addition to, the target gene sequence shownherein. In some embodiments, when the complement of a target gene isdetected, a polynucleotide for detection is used that is complementaryto the complement of the target gene. In some some embodiments, apolynucleotide for detection comprises at least a portion that is atleast 90%, at least 95%, or 100% identical in sequence to the targetgene, although it may comprise modified nucleotides.

4.2.4. Exemplary Analytical Methods

As described above, methods are presented for detecting Trichomonasvaginalis. The methods comprise detecting the presence of the TV 40Sribosomal protein (Tv40Srp) gene in a sample from a subject. In someembodiments, the method further comprises detecting at least oneendogenous control (such as an SAC) and/or at least one exogenouscontrol (such as an SPC). In some embodiments, detection of the TV 40Sribosomal protein (Tv40Srp) gene indicates the presence of TV, even ifthe endogenous control and/or exogenous control is not detected in theassay. In some embodiments, if the TV 40S ribosomal protein (Tv40Srp)gene is not detected, the result is considered to be negative for TVonly if the controls are detected. In some embodiments, if the TV 40Sribosomal protein (Tv40Srp) gene is not detected, the result isconsidered to be negative for TV only if the endogenous control andexogenous control are detected.

Any analytical procedure capable of permitting specific detection of atarget gene may be used in the methods herein presented. Exemplarynonlimiting analytical procedures include, but are not limited to,nucleic acid amplification methods, PCR methods, isothermalamplification methods, and other analytical detection methods known tothose skilled in the art.

In some embodiments, the method of detecting a target gene, such as theTV 40S ribosomal protein (Tv40Srp) gene, comprises amplifying the geneand/or a complement thereof. Such amplification can be accomplished byany method. Exemplary methods include, but are not limited to,isothermal amplification, real time PCR, endpoint PCR, and amplificationusing T7 polymerase from a T7 promoter annealed to a DNA, such asprovided by the SenseAmp Plus™ Kit available at Implen, Germany.

When a target gene is amplified, in some embodiments, an amplicon of thetarget gene is formed. An amplicon may be single stranded ordouble-stranded. In some embodiments, when an amplicon issingle-stranded, the sequence of the amplicon is related to the targetgene in either the sense or antisense orientation. In some embodiments,an amplicon of a target gene is detected rather than the target geneitself. Thus, when the methods discussed herein indicate that a targetgene is detected, such detection may be carried out on an amplicon ofthe target gene instead of, or in addition to, the target gene itself.In some embodiments, when the amplicon of the target gene is detectedrather than the target gene, a polynucleotide for detection is used thatis complementary to the complement of the target gene. In someembodiments, when the amplicon of the target gene is detected ratherthan the target gene, a polynucleotide for detection is used that iscomplementary to the target gene. Further, in some embodiments, multiplepolynucleotides for detection may be used, and some polynucleotides maybe complementary to the target gene and some polynucleotides may becomplementary to the complement of the target gene.

In some embodiments, the method of detecting the TV 40S ribosomalprotein (Tv40Srp) gene comprises PCR, as described below. In someembodiments, detecting one or more target genes comprises real-timemonitoring of a PCR reaction, which can be accomplished by any method.Such methods include, but are not limited to, the use of TaqMan®,molecular beacons, or Scorpion probes (i.e., energy transfer (ET)probes, such as FRET probes) and the use of intercalating dyes, such asSYBR green, EvaGreen, thiazole orange, YO-PRO, TO-PRO, etc.

Nonlimiting exemplary conditions for amplifying a target gene are asfollows. An exemplary cycle comprises an initial denaturation at 90° C.to 100° C. for 30 seconds to 5 minutes, followed by cycling thatcomprises denaturation at 90° C. to 100° C. for 1 to 10 seconds,followed by annealing and amplification at 60° C. to 75° C. for 10 to 30seconds. A further exemplary cycle comprises 1 minute at 95° C.,followed by up to 40 cycles of 5 seconds at 92.5° C., 20 seconds at 68°C. In some embodiments, for the first cycle following the initialdenaturation step, the cycle denaturation step is omitted. In someembodiments, Taq polymerase is used for amplification. In someembodiments, the cycle is carried out at least 10 times, at least 15times, at least 20 times, at least 25 times, at least 30 times, at least35 times, at least 40 times, or at least 45 times. In some embodiments,Taq is used with a hot start function. In some embodiments, theamplification reaction occurs in a GeneXpert® cartridge, andamplification of the TV 40S ribosomal protein (Tv40Srp) gene, anendogenous control, and an exogenous control occurs in the samereaction. In some embodiments, detection of the TV 40S ribosomal protein(Tv40Srp) gene occurs in less than 3 hours, less than 2.5 hours, lessthan 2 hours, less than 1 hour, or less than 30 minutes from initialdenaturation through the last extension.

In some embodiments, detection of a target gene comprises forming acomplex comprising a polynucleotide that is complementary to a targetgene or to a complement thereof, and a nucleic acid selected from thetarget gene, a DNA amplicon of the target gene, and a complement of thetarget gene. Thus, in some embodiments, the polynucleotide forms acomplex with a target gene. In some embodiments, the polynucleotideforms a complex with a complement of the target gene. In someembodiments, the polynucleotide forms a complex with a DNA amplicon ofthe target gene. When a double-stranded DNA amplicon is part of acomplex, as used herein, the complex may comprise one or both strands ofthe DNA amplicon. Thus, in some embodiments, a complex comprises onlyone strand of the DNA amplicon. In some embodiments, a complex is atriplex and comprises the polynucleotide and both strands of the DNAamplicon. In some embodiments, the complex is formed by hybridizationbetween the polynucleotide and the target gene, complement of the targetgene, or DNA amplicon of the target gene. The polynucleotide, in someembodiments, is a primer or probe.

In some embodiments, a method comprises detecting the complex. In someembodiments, the complex does not have to be associated at the time ofdetection. That is, in some embodiments, a complex is formed, thecomplex is then dissociated or destroyed in some manner, and componentsfrom the complex are detected. An example of such a system is a TaqMan®assay. In some embodiments, when the polynucleotide is a primer,detection of the complex may comprise amplification of the target gene,a complement of the target gene, or a DNA amplicon of the target gene.

In some embodiments the analytical method used for detecting at leastone target gene in the methods set forth herein includes real-timequantitative PCR. In some embodiments, the analytical method used fordetecting at least one target gene includes the use of a TaqMan® probe.The assay uses energy transfer (“ET”), such as fluorescence resonanceenergy transfer (“FRET”), to detect and quantitate the synthesized PCRproduct. Typically, the TaqMan® probe comprises a fluorescent dyemolecule coupled to the 5′-end and a quencher molecule coupled to the3′-end, such that the dye and the quencher are in close proximity,allowing the quencher to suppress the fluorescence signal of the dye viaFRET. When the polymerase replicates the chimeric amplicon template towhich the TaqMan® probe is bound, the 5′-nuclease of the polymerasecleaves the probe, decoupling the dye and the quencher so that the dyesignal (such as fluorescence) is detected. Signal (such as fluorescence)increases with each PCR cycle proportionally to the amount of probe thatis cleaved.

In some embodiments, a target gene is considered to be detected if anysignal is generated from the TaqMan probe during the PCR cycling. Forexample, in some embodiments, if the PCR includes 40 cycles, if a signalis generated at any cycle during the amplification, the target gene isconsidered to be present and detected. In some some embodiments, if nosignal is generated by the end of the PCR cycling, the target gene isconsidered to be absent and not detected.

In some embodiments, quantitation of the results of real-time PCR assaysis done by constructing a standard curve from a nucleic acid of knownconcentration and then extrapolating quantitative information for targetgenes of unknown concentration. In some embodiments, the nucleic acidused for generating a standard curve is a DNA (for example, anendogenous control, or an exogenous control). In some embodiments, thenucleic acid used for generating a standard curve is a purifieddouble-stranded plasmid DNA or a single-stranded DNA generated in vitro.

In some embodiments, in order for an assay to indicate that TV is notpresent in a sample, the Ct values for an endogenous control (such as anSAC) and/or an exogenous control (such as an SPC) must be within apreviously-determined valid range. That is, in some embodiments, theabsence of TV cannot be confirmed unless the controls are detected,indicating that the assay was successful. Ct values are inverselyproportional to the amount of nucleic acid target in a sample.

In some embodiments, a threshold Ct (or a “cutoff Ct”) value for atarget gene (including an endogenous control and/or exogenous control),below which the gene is considered to be detected, has previously beendetermined. In some embodiments, a threshold Ct is determined usingsubstantially the same assay conditions and system (such as aGeneXpert®) on which the samples will be tested.

In addition to the TagMan® assays, other real-time PCR chemistriesuseful for detecting and quantitating PCR products in the methodspresented herein include, but are not limited to, Molecular Beacons,Scorpion probes and intercalating dyes, such as SYBR Green, EvaGreen,thiazole orange, YO-PRO, TO-PRO, etc., which are discussed below.

In various embodiments, real-time PCR detection is utilized to detect,in a single multiplex reaction, the TV 40S ribosomal protein (Tv40Srp)gene, an endogenous control, and an exogenous control. In some multiplexembodiments, a plurality of probes, such as TaqMan® probes, eachspecific for a different target, is used. In some embodiments, eachtarget gene-specific probe is spectrally distinguishable from the otherprobes used in the same multiplex reaction.

Real-time PCR is performed using any PCR instrumentation available inthe art. Typically, instrumentation used in real-time PCR datacollection and analysis comprises a thermal cycler, optics forfluorescence excitation and emission collection, and optionally acomputer and data acquisition and analysis software.

In some embodiments, detection and/or quantitation of real-time PCRproducts is accomplished using a dye that binds to double-stranded DNAproducts, such as SYBR Green, EvaGreen, thiazole orange, YO-PRO, TO-PRO,etc. In some embodiments, the analytical method used in the methodsdescribed herein is a DASL® (DNA-mediated Annealing, Selection,Extension, and Ligation) Assay. In some embodiments, the analyticalmethod used for detecting and quantifying the target genes in themethods described herein is a bead-based flow cytometric assay. See LuJ. et al. (2005) Nature 435:834-838, which is incorporated herein byreference in its entirety. An example of a bead-based flow cytometricassay is the xMAP® technology of Luminex, Inc. Seewww.luminexcorp.com/technology/index.html. In some embodiments, theanalytical method used for detecting and quantifying the levels of theat least one target gene in the methods described herein is by gelelectrophoresis and detection with labeled probes (e.g., probes labeledwith a radioactive or chemiluminescent label), such as by northernblotting. In some embodiments, exemplary probes contain one or moreaffinity-enhancing nucleotide analogs as discussed below, such as lockednucleic acid (“LNA”) analogs, which contain a bicyclic sugar moietyinstead of deoxyribose or ribose sugars. See, e.g., Varallyay, E. et al.(2008) Nature Protocols 3(2):190-196, which is incorporated herein byreference in its entirety. In some embodiments, detection andquantification of one or more target genes is accomplished usingmicrofluidic devices and single-molecule detection.

Optionally, the sample DNA is modified before hybridization. The targetDNA/probe duplex is then passed through channels in a microfluidicdevice and that comprise detectors that record the unique signal of the3 labels. In this way, individual molecules are detected by their uniquesignal and counted. See U.S. Pat. Nos. 7,402,422 and 7,351,538 to Fuchset al., U.S. Genomics, Inc., each of which is incorporated herein byreference in its entirety.

4.2.5. Exemplary Automation and Systems

In some embodiments, gene expression is detected using an automatedsample handling and/or analysis platform. In some embodiments,commercially available automated analysis platforms are utilized. Forexample, in some embodiments, the GeneXpert® system (Cepheid, Sunnyvale,Calif.) is utilized.

The present invention is illustrated for use with the GeneXpert system.Exemplary sample preparation and analysis methods are described below.However, the present invention is not limited to a particular detectionmethod or analysis platform. One of skill in the art recognizes that anynumber of platforms and methods may be utilized.

The GeneXpert® utilizes a self-contained, single use cartridge. Sampleextraction, amplification, and detection may all carried out within thisself-contained “laboratory in a cartridge.” (See e.g., U.S. Pat. Nos.5,958,349, 6,403,037, 6,440,725, 6,783,736, 6,818,185; each of which isherein incorporated by reference in its entirety.)

Components of the cartridge include, but are not limited to, processingchambers containing reagents, filters, and capture technologies usefulto extract, purify, and amplify target nucleic acids. A valve enablesfluid transfer from chamber to chamber and contain nucleic acids lysisand filtration components. An optical window enables real-time opticaldetection. A reaction tube enables very rapid thermal cycling.

In some embodiments, the GenXpert® system includes a plurality ofmodules for scalability. Each module includes a plurality of cartridges,along with sample handling and analysis components.

After the sample is added to the cartridge, the sample is contacted withlysis buffer and released DNA is bound to a DNA-binding substrate suchas a silica or glass substrate. The sample supernatant is then removedand the DNA eluted in an elution buffer such as a Tris/EDTA buffer. Theeluate may then be processed in the cartridge to detect target genes asdescribed herein. In some embodiments, the eluate is used toreconstitute at least some of the PCR reagents, which are present in thecartridge as lyophilized particles.

In some embodiments, PCR is used to amplify and analyze the presence ofthe target genes. In some embodiments, the PCR uses Taq polymerase withhot start function, such as AptaTaq (Roche). In some embodiments, theinitial denaturation is at 90° C. to 100° C. for 30 seconds to 5minutes; the cycling denaturation temperature is 90° C. to 100° C. for 1to 10 seconds; the cycling anneal and amplification temperature is 60°C. to 75° C. for 10 to 30 seconds; and up to 50 cycles are performed.

In some embodiments, a double-denature method is used to amplify lowcopy number targets. A double-denature method comprises, in someembodiments, a first denaturation step followed by addition of primersand/or probes for detecting target genes. All or a substantial portionof the DNA-containing sample (such as a DNA eluate) is then denatured asecond time before, in some instances, a portion of the sample isaliquotted for cycling and detection of the target genes. While notintending to be bound by any particular theory, the double-denatureprotocol may increase the chances that a low copy number target gene (orits complement) will be present in the aliquot selected for cycling anddetection because the second denaturation effectively doubles the numberof targets (i.e., it separates the target and its complement into twoseparate templates) before an aliquot is selected for cycling. In someembodiments, the first denaturation step comprises heating to atemperature of 90° C. to 100° C. for a total time of 30 seconds to 5minutes. In some embodiments, the second denaturation step comprisesheating to a temperature of 90° C. to 100° C. for a total time of 5seconds to 3 minutes. In some embodiments, the first denaturation stepand/or the second denaturation step is carried out by heating aliquotsof the sample separately. In some embodiments, each aliquot may beheated for the times listed above. As a non-limiting example, a firstdenaturation step for a DNA-containing sample (such as a DNA eluate) maycomprise heating at least one, at least two, at least three, or at leastfour aliquots of the sample separately (either sequentially orsimultaneously) to a temperature of 90° C. to 100° C. for 60 secondseach. As a non-limiting example, a second denaturation step for aDNA-containing sample (such as a DNA eluate) containing enzyme, primers,and probes may comprise heating at least one, at least two, at leastthree, or at least four aliquots of the eluate separately (eithersequentially or simultaneously) to a temperature of 90° C. to 100° C.for 5 seconds each. In some embodiments, an aliquot is the entireDNA-containing sample (such as a DNA eluate). In some embodiments, analiquot is less than the entire DNA-containing sample (such as a DNAeluate).

In some embodiments, target genes in a DNA-containing sample, such as aDNA eluate, are detected using the following protocol: One or morealiquots of the DNA-containing sample are heated separately to 95° C.for 60 seconds each. The enzyme and primers and probes are added to theDNA-containing sample and one or more aliquots are heated separately to95° C. for 5 seconds each. At least one aliquot of the DNA-containingsample containing enzyme, primers, and probes is then heated to 94° C.for 60 seconds. The aliquot is then cycled 45 times with the following2-step cycle: (1) 94° C. for 5 seconds, (2) 66° C. for 30 seconds.

The present invention is not limited to particular primer and/or probesequences. Exemplary amplification primers and detection probes aredescribed in the Examples.

In some embodiments, an off-line centrifugation is used, for example,with samples with low cellular content. The sample, with or without abuffer added, is centrifuged and the supernatant removed. The pellet isthen resuspended in a smaller volume of either supernatant or thebuffer. The resuspended pellet is then analyzed as described herein.

4.2.6. Exemplary Data Analysis

In some embodiments, the presence of TV is detected if the Ct value forthe TV 40S ribosomal protein (Tv40Srp) gene is below a certainthreshold. In some embodiments the valid range of Ct values is 9 to 39.9Ct. In some such embodiments, if no amplification above background isobserved from the TV-specific primers after 40 cycles, the sample isconsidered to be negative for TV.

In some embodiments, a computer-based analysis program is used totranslate the raw data generated by the detection assay into data ofpredictive value for a clinician. The clinician can access thepredictive data using any suitable means. Thus, in some embodiments, thepresent invention provides the further benefit that the clinician, whois not likely to be trained in genetics or molecular biology, need notunderstand the raw data. The data is presented directly to the clinicianin its most useful form. The clinician is then able to immediatelyutilize the information in order to optimize the care of the subject.

The present invention contemplates any method capable of receiving,processing, and transmitting the information to and from laboratoriesconducting the assays, information provides, medical personal, andsubjects. For example, in some embodiments of the present invention, asample (e.g., a biopsy or a serum or urine sample) is obtained from asubject and submitted to a profiling service (e.g., clinical lab at amedical facility, genomic profiling business, etc.), located in any partof the world (e.g., in a country different than the country where thesubject resides or where the information is ultimately used) to generateraw data. Where the sample comprises a tissue or other biologicalsample, the subject may visit a medical center to have the sampleobtained and sent to the profiling center, or subjects may collect thesample themselves (e.g., a urine sample) and directly send it to aprofiling center. Where the sample comprises previously determinedbiological information, the information may be directly sent to theprofiling service by the subject (e.g., an information card containingthe information may be scanned by a computer and the data transmitted toa computer of the profiling center using an electronic communicationsystems). Once received by the profiling service, the sample isprocessed and a profile is produced (i.e., expression data), specificfor the diagnostic or prognostic information desired for the subject.

The profile data is then prepared in a format suitable forinterpretation by a treating clinician. For example, rather thanproviding raw expression data, the prepared format may represent adiagnosis or risk assessment (e.g., presence of TV) for the subject,with or without recommendations for particular treatment options. Thedata may be displayed to the clinician by any suitable method. Forexample, in some embodiments, the profiling service generates a reportthat can be printed for the clinician (e.g., at the point of care) ordisplayed to the clinician on a computer monitor.

In some embodiments, the information is first analyzed at the point ofcare or at a regional facility. The raw data is then sent to a centralprocessing facility for further analysis and/or to convert the raw datato information useful for a clinician or patient. The central processingfacility provides the advantage of privacy (all data is stored in acentral facility with uniform security protocols), speed, and uniformityof data analysis. The central processing facility can then control thefate of the data following treatment of the subject. For example, usingan electronic communication system, the central facility can providedata to the clinician, the subject, or researchers.

In some embodiments, the subject is able to directly access the datausing the electronic communication system. The subject may chose furtherintervention or counseling based on the results. In some embodiments,the data is used for research use. For example, the data may be used tofurther optimize the inclusion or elimination of markers as usefulindicators of a particular condition or stage of disease or as acompanion diagnostic to determine a treatment course of action.

4.2.7. Exemplary Polynucleotides

In some embodiments, polynucleotides are provided. In some embodiments,synthetic polynucleotides are provided. Synthetic polynucleotides, asused herein, refer to polynucleotides that have been synthesized invitro either chemically or enzymatically. Chemical synthesis ofpolynucleotides includes, but is not limited to, synthesis usingpolynucleotide synthesizers, such as OligoPilot (GE Healthcare), ABI3900 DNA Synthesizer (Applied Biosystems), and the like. Enzymaticsynthesis includes, but is not limited, to producing polynucleotides byenzymatic amplification, e.g., PCR. A polynucleotide may comprise one ormore nucleotide analogs (i.e., modified nucleotides) discussed herein.

In some embodiments, a polynucleotide is provided that comprises aregion that is at least 90%, at least 95%, or 100% identical to, or atleast 90%, at least 95%, or 100% complementary to, at least 8, at least9, at least 10, at least 11, at least 12, at least 13, at least 14, atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, at elast 21, at least 22, at least 23, at least 24, at least 25, atleast 26, at least 27, at least 28, at least 29, or at least 30contiguous nucleotides of the TV 40S ribosomal protein (Tv40Srp) gene.In some embodiments, a polynucleotide is provided that comprises aregion that is at least 90%, at least 95%, or 100% identical to, orcomplementary to, a span of 6 to 100, 8 to 100, 8 to 75, 8 to 50, 8 to40, or 8 to 30 contiguous nucleotides of the TV 40S ribosomal protein(Tv40Srp) gene. Nonlimiting exemplary polynucleotides are shown in Table1.

In various embodiments, a polynucleotide comprises fewer than 500, fewerthan 300, fewer than 200, fewer than 150, fewer than 100, fewer than 75,fewer than 50, fewer than 40, or fewer than 30 nucleotides. In variousembodiments, a polynucleotide is between 6 and 200, between 8 and 200,between 8 and 150, between 8 and 100, between 8 and 75, between 8 and50, between 8 and 40, between 8 and 30, between 15 and 100, between 15and 75, between 15 and 50, between 15 and 40, or between 15 and 30nucleotides long.

In some embodiments, the polynucleotide is a primer. In someembodiments, the primer is labeled with a detectable moiety. In someembodiments, a primer is not labeled. A primer, as used herein, is apolynucleotide that is capable of selectively hybridizing to a targetgene or to an amplicon that has been amplified from a target gene(collectively referred to as “template”), and, in the presence of thetemplate, a polymerase and suitable buffers and reagents, can beextended to form a primer extension product.

In some embodiments, the polynucleotide is a probe. In some embodiments,the probe is labeled with a detectable moiety. A detectable moiety, asused herein, includes both directly detectable moieties, such asfluorescent dyes, and indirectly detectable moieties, such as members ofbinding pairs. When the detectable moiety is a member of a binding pair,in some embodiments, the probe can be detectable by incubating the probewith a detectable label bound to the second member of the binding pair.In some embodiments, a probe is not labeled, such as when a probe is acapture probe, e.g., on a microarray or bead. In some embodiments, aprobe is not extendable, e.g., by a polymerase. In other embodiments, aprobe is extendable.

In some embodiments, the polynucleotide is a FRET probe that in someembodiments is labeled at the 5′-end with a fluorescent dye (donor) andat the 3′-end with a quencher (acceptor), a chemical group that absorbs(i.e., suppresses) fluorescence emission from the dye when the groupsare in close proximity (i.e., attached to the same probe). Thus, in someembodiments, the emission spectrum of the dye should overlapconsiderably with the absorption spectrum of the quencher. In otherembodiments, the dye and quencher are not at the ends of the FRET probe.

4.2.7.1. Exemplary Polynucleotide Modifications

In some embodiments, the methods of detecting at least one target genedescribed herein employ one or more polynucleotides that have beenmodified, such as polynucleotides comprising one or moreaffinity-enhancing nucleotide analogs. Modified polynucleotides usefulin the methods described herein include primers for reversetranscription, PCR amplification primers, and probes. In someembodiments, the incorporation of affinity-enhancing nucleotidesincreases the binding affinity and specificity of a polynucleotide forits target nucleic acid as compared to polynucleotides that contain onlydeoxyribonucleotides, and allows for the use of shorter polynucleotidesor for shorter regions of complementarity between the polynucleotide andthe target nucleic acid.

In some embodiments, affinity-enhancing nucleotide analogs includenucleotides comprising one or more base modifications, sugarmodifications and/or backbone modifications.

In some embodiments, modified bases for use in affinity-enhancingnucleotide analogs include 5-methylcytosine, isocytosine,pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine,2-aminopurine, inosine, diaminopurine, 2-chloro-6-aminopurine, xanthineand hypoxanthine.

In some embodiments, affinity-enhancing nucleotide analogs includenucleotides having modified sugars such as 2′-substituted sugars, suchas 2′-O-alkyl-ribose sugars, 2′-amino-deoxyribose sugars,2′-fluoro-deoxyribose sugars, 2′-fluoro-arabinose sugars, and2′-O-methoxyethyl-ribose (2′MOE) sugars. In some embodiments, modifiedsugars are arabinose sugars, or d-arabino-hexitol sugars.

In some embodiments, affinity-enhancing nucleotide analogs includebackbone modifications such as the use of peptide nucleic acids (PNA;e.g., an oligomer including nucleobases linked together by an amino acidbackbone). Other backbone modifications include phosphorothioatelinkages, phosphodiester modified nucleic acids, combinations ofphosphodiester and phosphorothioate nucleic acid, methylphosphonate,alkylphosphonates, phosphate esters, alkylphosphonothioates,phosphoramidates, carbamates, carbonates, phosphate triesters,acetamidates, carboxymethyl esters, methylphosphorothioate,phosphorodithioate, p-ethoxy, and combinations thereof.

In some embodiments, a polynucleotide includes at least oneaffinity-enhancing nucleotide analog that has a modified base, at leastnucleotide (which may be the same nucleotide) that has a modified sugar,and/or at least one internucleotide linkage that is non-naturallyoccurring.

In some embodiments, an affinity-enhancing nucleotide analog contains alocked nucleic acid (“LNA”) sugar, which is a bicyclic sugar. In someembodiments, a polynucleotide for use in the methods described hereincomprises one or more nucleotides having an LNA sugar. In someembodiments, a polynucleotide contains one or more regions consisting ofnucleotides with LNA sugars. In other embodiments, a polynucleotidecontains nucleotides with LNA sugars interspersed withdeoxyribonucleotides. See, e.g., Frieden, M. et al. (2008) Curr. Pharm.Des. 14(11):1138-1142.

4.2.7.2. Exemplary Primers

In some embodiments, a primer is provided. In some embodiments, a primeris at least 90%, at least 95%, or 100% identical to, or at least 90%, atleast 95%, or 100% complementary to, at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, at least 20, at elast21, at least 22, at least 23, at least 24, at least 25, at least 26, atleast 27, at least 28, at least 29, or at least 30 contiguousnucleotides of the TV 40S ribosomal protein (Tv40Srp) gene. In someembodiments, a primer is provided that comprises a region that is atleast 90%, at least 95%, or 100% identical to, or complementary to, aspan of 6 to 100, 8 to 100, 8 to 75, 8 to 50, 8 to 40, or 8 to 30contiguous nucleotides of the TV 40S ribosomal protein (Tv40Srp) gene.Nonlimiting exemplary primers are shown in Table 1. In some embodiments,a primer may also comprise portions or regions that are not identical orcomplementary to the target gene. In some embodiments, a region of aprimer that is at least 90%, at least 95%, or 100% identical orcomplementary to a target gene is contiguous, such that any region of aprimer that is not identical or complementary to the target gene doesnot disrupt the identical or complementary region.

In some embodiments, a primer comprises a portion that is at least 90%,at least 95%, or 100% identical to a region of a target gene. In somesuch embodiments, a primer that comprises a region that is at least 90%,at least 95%, or 100% identical to a region of the target gene iscapable of selectively hybridizing to an amplicon that has been producedby amplification of the target gene. In some embodiments, the primer iscomplementary to a sufficient portion of the amplicon such that itselectively hybridizes to the amplicon under the conditions of theparticular assay being used.

As used herein, “selectively hybridize” means that a polynucleotide,such as a primer or probe, will hybridize to a particular nucleic acidin a sample with at least 5-fold greater affinity than it will hybridizeto another nucleic acid present in the same sample that has a differentnucleotide sequence in the hybridizing region. Exemplary hybridizationconditions are discussed herein, for example, in the context of areverse transcription reaction or a PCR amplification reaction. In someembodiments, a polynucleotide will hybridize to a particular nucleicacid in a sample with at least 10-fold greater affinity than it willhybridize to another nucleic acid present in the same sample that has adifferent nucleotide sequence in the hybridizing region.

In some embodiments, a primer comprises a detectable moiety.

In some embodiments, primer pairs are provided. Such primer pairs aredesigned to amplify a portion of a target gene, such as the TV 40Sribosomal protein (Tv40Srp) gene, or an endogenous control such as asample adequacy control (SAC), or an exogenous control such as a sampleprocessing control (SPC). In some embodiments, a primer pair is designedto produce an amplicon that is 50 to 1500 nucleotides long, 50 to 1000nucleotides long, 50 to 750 nucleotides long, 50 to 500 nucleotideslong, 50 to 400 nucleotides long, 50 to 300 nucleotides long, 50 to 200nucleotides long, 50 to 150 nucleotides long, 100 to 300 nucleotideslong, 100 to 200 nucleotides long, or 100 to 150 nucleotides long.Nonlimiting exemplary primer pairs are shown in Table 1.

4.2.7.3. Exemplary Probes

In various embodiments, methods of detecting the presence of Trichomonasvaginalis comprise hybridizing nucleic acids of a sample with a probe.In some embodiments, the probe comprises a portion that is complementaryto a target gene, such as the TV 40S ribosomal protein (Tv40Srp) gene,or an endogenous control such as a sample adequacy control (SAC), or anexogenous control such as a sample processing control (SPC). In someembodiments, the probe comprises a portion that is at least 90%, atleast 95%, or 100% identical to a region of the target gene. In somesuch embodiments, a probe that is at least 90%, at least 95%, or 100%complementary to a target gene is complementary to a sufficient portionof the target gene such that it selectively hybridizes to the targetgene under the conditions of the particular assay being used. In someembodiments, a probe that is complementary to a target gene comprises aregion that is at least 90%, at least 95%, or 100% complementary to atleast 8, at least 9, at least 10, at least 11, at least 12, at least 13,at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25, at least 26, at least 27, at least 28, at least 29, orat least 30 contiguous nucleotides of the target gene. Nonlimitingexemplary probes are shown in Table 1. A probe that is at least 90%, atleast 95%, or 100% complementary to a target gene may also compriseportions or regions that are not complementary to the target gene. Insome embodiments, a region of a probe that is at least 90%, at least95%, or 100% complementary to a target gene is contiguous, such that anyregion of a probe that is not complementary to the target gene does notdisrupt the complementary region.

In some embodiments, the probe comprises a portion that is at least 90%,at least 95%, or 100% identical to a region of the target gene, such asthe TV 40S ribosomal protein (Tv40Srp) gene, or an endogenous controlsuch as a sample adequacy control (SAC), or an exogenous control such asa sample processing control (SPC). In some such embodiments, a probethat comprises a region that is at least 90%, at least 95%, or 100%identical to a region of the target gene is capable of selectivelyhybridizing to an amplicon that has been produced by amplification ofthe target gene. In some embodiments, the probe is at least 90%, atleast 95%, or 100% complementary to a sufficient portion of the ampliconsuch that it selectively hybridizes to the amplicon under the conditionsof the particular assay being used. In some embodiments, a probe that iscomplementary to a amplicon comprises a region that is at least 90%, atleast 95%, or 100% complementary to at least 8, at least 9, at least 10,at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, at least 20, at least21, at least 22, at least 23, at least 24, at least 25, at least 26, atleast 27, at least 28, at least 29, or at least 30 contiguousnucleotides of the amplicon. A probe that is at least 90%, at least 95%,or 100% complementary to an amplicon may also comprise portions orregions that are not complementary to the amplicon. In some embodiments,a region of a probe that is at least 90%, at least 95%, or 100%complementary to an amplicon is contiguous, such that any region of aprobe that is not complementary to the amplicon does not disrupt thecomplementary region.

In some embodiments, the method of detecting one or more target genescomprises: (a) amplifying a region of the target gene; and (b) detectingthe amplified region using real time PCR and a detection probe (whichmay be simultaneous with the amplification step (a)).

As described above, in some embodiments, real time PCR detection may beperformed using a FRET probe, which includes, but is not limited to, aTaqMan® probe, a Molecular beacon probe and a Scorpion probe. In someembodiments, the real time PCR detection is performed with a TaqMan®probe, i.e., a linear probe that typically has a fluorescent dyecovalently bound at one end of the DNA and a quencher moleculecovalently bound elsewhere, such as at the other end of, the DNA. TheFRET probe comprises a sequence that is complementary to a region of theamplicon such that, when the FRET probe is hybridized to the amplicon,the dye fluorescence is quenched, and when the probe is digested duringamplification of the amplicon, the dye is released from the probe andproduces a fluorescence signal. In some embodiments, the amount oftarget gene in the sample is proportional to the amount of fluorescencemeasured during amplification.

The TaqMan® probe typically comprises a region of contiguous nucleotideshaving a sequence that is at least 90%, at least 95%, or 100% identicalor complementary to a region of a target gene such that the probe isselectively hybridizable to a PCR amplicon of a region of the targetgene. In some embodiments, the probe comprises a region of at least 6contiguous nucleotides having a sequence that is fully complementary toor identically present in a region of a target gene. In someembodiments, the probe comprises a region that is at least 90%, at least95%, or 100% identical or complementary to at least 8 contiguousnucleotides, at least 10 contiguous nucleotides, at least 12 contiguousnucleotides, at least 14 contiguous nucleotides, or at least 16contiguous nucleotides of a target gene to be detected.

In some embodiments, the region of the amplicon that has a sequence thatis at least 90%, at least 95%, or 100% complementary to the TaqMan®probe sequence is at or near the center of the amplicon molecule. Insome embodiments, there are independently at least 2 nucleotides, suchas at least 3 nucleotides, such as at least 4 nucleotides, such as atleast 5 nucleotides of the amplicon at the 5′-end and at the 3′-end ofthe region of complementarity.

In some embodiments, Molecular Beacons can be used to detect PCRproducts. Like TaqMan® probes, Molecular Beacons use FRET to detect aPCR product via a probe having a fluorescent dye and a quencher attachedat the ends of the probe. Unlike TaqMan® probes, Molecular Beaconsremain intact during the PCR cycles. Molecular Beacon probes form astem-loop structure when free in solution, thereby allowing the dye andquencher to be in close enough proximity to cause fluorescencequenching. When the Molecular Beacon hybridizes to a target, thestem-loop structure is abolished so that the dye and the quencher becomeseparated in space and the dye fluoresces. Molecular Beacons areavailable, e.g., from Gene Link™ (seewww.genelink.com/newsite/products/mbintro.asp).

In some embodiments, Scorpion probes can be used as bothsequence-specific primers and for PCR product detection. Like MolecularBeacons, Scorpion probes form a stem-loop structure when not hybridizedto a target nucleic acid. However, unlike Molecular Beacons, a Scorpionprobe achieves both sequence-specific priming and PCR product detection.A fluorescent dye molecule is attached to the 5′-end of the Scorpionprobe, and a quencher is attached elsewhere, such as to the 3′-end. The3′ portion of the probe is complementary to the extension product of thePCR primer, and this complementary portion is linked to the 5′-end ofthe probe by a non-amplifiable moiety. After the Scorpion primer isextended, the target-specific sequence of the probe binds to itscomplement within the extended amplicon, thus opening up the stem-loopstructure and allowing the dye on the 5′-end to fluoresce and generate asignal. Scorpion probes are available from, e.g, Premier BiosoftInternational (see www.premierbiosoft.com/tech_notes/Scorpion.html).

In some embodiments, labels that can be used on the FRET probes includecolorimetric and fluorescent dyes such as Alexa Fluor dyes, BODIPY dyes,such as BODIPY FL; Cascade Blue; Cascade Yellow; coumarin and itsderivatives, such as 7-amino-4-methylcoumarin, aminocoumarin andhydroxycoumarin; cyanine dyes, such as Cy3 and Cy5; eosins anderythrosins; fluorescein and its derivatives, such as fluoresceinisothiocyanate; macrocyclic chelates of lanthanide ions, such as QuantumDye™; Marina Blue; Oregon Green; rhodamine dyes, such as rhodamine red,tetramethylrhodamine and rhodamine 6G; Texas Red; fluorescent energytransfer dyes, such as thiazole orange-ethidium heterodimer; and, TOTAB.

Specific examples of dyes include, but are not limited to, thoseidentified above and the following: Alexa Fluor 350, Alexa Fluor 405,Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500. Alexa Fluor 514,Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568,Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647,Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, and, Alexa Fluor 750;amine-reactive BODIPY dyes, such as BODIPY 493/503, BODIPY 530/550,BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY630/650, BODIPY 650/655, BODIPY FL, BODIPY R6G, BODIPY TMR, and,BODIPY-TR; Cy3, Cy5, 6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE,Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG,Rhodamine Green, Rhodamine Red, Renographin, ROX, SYPRO, TAMRA,2′,4′,5′,7′-Tetrabromosulfonefluorescein, and TET.

Examples of dye/quencher pairs (i.e., donor/acceptor pairs) include, butare not limited to, fluorescein/tetramethylrhodamine;IAEDANS/fluorescein; EDANS/dabcyl; fluorescein/fluorescein; BODIPYFL/BODIPY FL; fluorescein/QSY 7 or QSY 9 dyes. When the donor andacceptor are the same, FRET may be detected, in some embodiments, byfluorescence depolarization. Certain specific examples of dye/quencherpairs (i.e., donor/acceptor pairs) include, but are not limited to,Alexa Fluor 350/Alexa Fluor488; Alexa Fluor 488/Alexa Fluor 546; AlexaFluor 488/Alexa Fluor 555; Alexa Fluor 488/Alexa Fluor 568; Alexa Fluor488/Alexa Fluor 594; Alexa Fluor 488/Alexa Fluor 647; Alexa Fluor546/Alexa Fluor 568; Alexa Fluor 546/Alexa Fluor 594; Alexa Fluor546/Alexa Fluor 647; Alexa Fluor 555/Alexa Fluor 594; Alexa Fluor555/Alexa Fluor 647; Alexa Fluor 568/Alexa Fluor 647; Alexa Fluor594/Alexa Fluor 647; Alexa Fluor 350/QSY35; Alexa Fluor 350/dabcyl;Alexa Fluor 488/QSY 35; Alexa Fluor 488/dabcyl; Alexa Fluor 488/QSY 7 orQSY 9; Alexa Fluor 555/QSY 7 or QSY9; Alexa Fluor 568/QSY 7 or QSY 9;Alexa Fluor 568/QSY 21; Alexa Fluor 594/QSY 21; and Alexa Fluor 647/QSY21. In some instances, the same quencher may be used for multiple dyes,for example, a broad spectrum quencher, such as an Iowa Black® quencher(Integrated DNA Technologies, Coralville, Iowa) or a Black HoleQuencher™ (BHQ™; Sigma-Aldrich, St. Louis, Mo.).

In some embodiments, for example, in a multiplex reaction in which twoor more moieties (such as amplicons) are detected simultaneously, eachprobe comprises a detectably different dye such that the dyes may bedistinguished when detected simultaneously in the same reaction. Oneskilled in the art can select a set of detectably different dyes for usein a multiplex reaction.

Specific examples of fluorescently labeled ribonucleotides useful in thepreparation of PCR probes for use in some embodiments of the methodsdescribed herein are available from Molecular Probes (Invitrogen), andthese include, Alexa Fluor 488-5-UTP, Fluorescein-12-UTP, BODIPYFL-14-UTP, BODIPY TMR-14-UTP, Tetramethylrhodamine-6-UTP, Alexa Fluor546-14-UTP, Texas Red-5-UTP, and BODIPY TR-14-UTP. Other fluorescentribonucleotides are available from Amersham Biosciences (GE Healthcare),such as Cy3-UTP and Cy5-UTP.

Examples of fluorescently labeled deoxyribonucleotides useful in thepreparation of PCR probes for use in the methods described hereininclude Dinitrophenyl (DNP)-1′-dUTP, Cascade Blue-7-dUTP, Alexa Fluor488-5-dUTP, Fluorescein-12-dUTP, Oregon Green 488-5-dUTP, BODIPYFL-14-dUTP, Rhodamine Green-5-dUTP, Alexa Fluor 532-5-dUTP, BODIPYTMR-14-dUTP, Tetramethylrhodamine-6-dUTP, Alexa Fluor 546-14-dUTP, AlexaFluor 568-5-dUTP, Texas Red-12-dUTP, Texas Red-5-dUTP, BODIPYTR-14-dUTP, Alexa Fluor 594-5-dUTP, BODIPY 630/650-14-dUTP, BODIPY650/665-14-dUTP; Alexa Fluor 488-7-OBEA-dCTP, Alexa Fluor546-16-OBEA-dCTP, Alexa Fluor 594-7-OBEA-dCTP, Alexa Fluor647-12-OBEA-dCTP. Fluorescently labeled nucleotides are commerciallyavailable and can be purchased from, e.g., Invitrogen.

In some embodiments, dyes and other moieties, such as quenchers, areintroduced into polynucleotide used in the methods described herein,such as FRET probes, via modified nucleotides. A “modified nucleotide”refers to a nucleotide that has been chemically modified, but stillfunctions as a nucleotide. In some embodiments, the modified nucleotidehas a chemical moiety, such as a dye or quencher, covalently attached,and can be introduced into a polynucleotide, for example, by way ofsolid phase synthesis of the polynucleotide. In other embodiments, themodified nucleotide includes one or more reactive groups that can reactwith a dye or quencher before, during, or after incorporation of themodified nucleotide into the nucleic acid. In specific embodiments, themodified nucleotide is an amine-modified nucleotide, i.e., a nucleotidethat has been modified to have a reactive amine group. In someembodiments, the modified nucleotide comprises a modified base moiety,such as uridine, adenosine, guanosine, and/or cytosine. In specificembodiments, the amine-modified nucleotide is selected from5-(3-aminoallyl)-UTP; 8-[(4-amino)butyl]-amino-ATP and8-[(6-amino)butyl]-amino-ATP; N6-(4-amino)butyl-ATP,N6-(6-amino)butyl-ATP, N4-[2,2-oxy-bis-(ethylamine)]-CTP;N6-(6-Amino)hexyl-ATP; 8-[(6-Amino)hexyl]-amino-ATP;5-propargylamino-CTP, 5-propargylamino-UTP. In some embodiments,nucleotides with different nucleobase moieties are similarly modified,for example, 5-(3-aminoallyl)-GTP instead of 5-(3-aminoallyl)-UTP. Manyamine modified nucleotides are commercially available from, e.g.,Applied Biosystems, Sigma, Jena Bioscience and TriLink.

Exemplary detectable moieties also include, but are not limited to,members of binding pairs. In some such embodiments, a first member of abinding pair is linked to a polynucleotide. The second member of thebinding pair is linked to a detectable label, such as a fluorescentlabel. When the polynucleotide linked to the first member of the bindingpair is incubated with the second member of the binding pair linked tothe detectable label, the first and second members of the binding pairassociate and the polynucleotide can be detected. Exemplary bindingpairs include, but are not limited to, biotin and streptavidin,antibodies and antigens, etc.

In some embodiments, multiple target genes are detected in a singlemultiplex reaction. In some such embodiments, each probe that istargeted to a unique amplicon is spectrally distinguishable whenreleased from the probe, in which case each target gene is detected by aunique fluorescence signal. In some embodiments, two or more targetgenes are detected using the same fluorescent signal, in which casedetection of that signal indicates the presence of either of the targetgenes or both.

One skilled in the art can select a suitable detection method for aselected assay, e.g., a real-time PCR assay. The selected detectionmethod need not be a method described above, and may be any method.

4.3. Exemplary Compositions and Kits

In another aspect, compositions are provided. In some embodiments,compositions are provided for use in the methods described herein.

In some embodiments, compositions are provided that comprise at leastone target gene-specific primer. The term “target gene-specific primer”encompasses primers that have a region of contiguous nucleotides havinga sequence that is (i) at least 90%, at least 95%, or 100% identical toa region of a target gene, or (ii) at least 90%, at least 95%, or 100%complementary to the sequence of a region of contiguous nucleotidesfound in a target gene. In some embodiments, a composition is providedthat comprises at least one pair of target gene-specific primers. Theterm “pair of target gene-specific primers” encompasses pairs of primersthat are suitable for amplifying a defined region of a target gene. Apair of target gene-specific primers typically comprises a first primerthat comprises a sequence that is at least 90%, at least 95%, or 100%identical to the sequence of a region of a target gene and a secondprimer that comprises a sequence that is at least 90%, at least 95%, or100% complementary to a region of a target gene. A pair of primers istypically suitable for amplifying a region of a target gene that is 50to 1500 nucleotides long, 50 to 1000 nucleotides long, 50 to 750nucleotides long, 50 to 500 nucleotides long, 50 to 400 nucleotideslong, 50 to 300 nucleotides long, 50 to 200 nucleotides long, 50 to 150nucleotides long, 100 to 300 nucleotides long, 100 to 200 nucleotideslong, or 100 to 150 nucleotides long. Nonlimiting exemplary primers, andpairs of primers, are shown in Table 1.

In some embodiments, a composition comprises at least one pair of targetgene-specific primers. In some embodiments, a composition additionallycomprises a pair of target gene-specific primers for amplifying anendogenous control (such as an SAC) and/or one pair of targetgene-specific primers for amplifying an exogenous control (such as anSPC).

In some embodiments, a composition comprises at least one targetgene-specific probe. The term “target gene-specific probe” encompassesprobes that have a region of contiguous nucleotides having a sequencethat is (i) at least 90%, at least 95%, or 100% identical to a region ofa target gene, or (ii) at least 90%, at least 95%, or 100% complementaryto the sequence of a region of contiguous nucleotides found in a targetgene. Nonlimiting exemplary target-specific probes are shown in Table 1.

In some embodiments, a composition (including a composition describedabove that comprises one or more pairs of target gene-specific primers)comprises one or more probes for detecting the target genes. In someembodiments, a composition comprises a probe for detecting an endogenouscontrol (such as an SAC) and/or a probe for detecting an exogenouscontrol (such as an SPC).

In some embodiments, a composition is an aqueous composition. In someembodiments, the aqueous composition comprises a buffering component,such as phosphate, tris, HEPES, etc., and/or additional components, asdiscussed below. In some embodiments, a composition is dry, for example,lyophilized, and suitable for reconstitution by addition of fluid. A drycomposition may include one or more buffering components and/oradditional components.

In some embodiments, a composition further comprises one or moreadditional components. Additional components include, but are notlimited to, salts, such as NaCl, KCl, and MgCl₂; polymerases, includingthermostable polymerases such as Taq; dNTPs; bovine serum albumin (BSA)and the like; reducing agents, such as β-mercaptoethanol; EDTA and thelike; etc. One skilled in the art can select suitable compositioncomponents depending on the intended use of the composition.

In some embodiments, compositions are provided that comprise at leastone polynucleotide for detecting at least one target gene. In someembodiments, the polynucleotide is used as a primer for a reversetranscriptase reaction. In some embodiments, the polynucleotide is usedas a primer for amplification. In some embodiments, the polynucleotideis used as a primer for PCR. In some embodiments, the polynucleotide isused as a probe for detecting at least one target gene. In someembodiments, the polynucleotide is detectably labeled. In someembodiments, the polynucleotide is a FRET probe. In some embodiments,the polynucleotide is a TaqMan® probe, a Molecular Beacon, or a Scorpionprobe.

In some embodiments, a composition comprises at least one FRET probehaving a sequence that is at least 90%, at least 95%, or 100% identical,or at least 90%, at least 95%, or 100% complementary, to a region of,the TV 40S ribosomal protein (Tv40Srp) gene. In some embodiments, a FRETprobe is labeled with a donor/acceptor pair such that when the probe isdigested during the PCR reaction, it produces a unique fluorescenceemission that is associated with a specific target gene. In someembodiments, when a composition comprises multiple FRET probes, eachprobe is labeled with a different donor/acceptor pair such that when theprobe is digested during the PCR reaction, each one produces a uniquefluorescence emission that is associated with a specific probe sequenceand/or target gene. In some embodiments, the sequence of the FRET probeis complementary to a target region of a target gene. In otherembodiments, the FRET probe has a sequence that comprises one or morebase mismatches when compared to the sequence of the best-aligned targetregion of a target gene.

In some embodiments, a composition comprises a FRET probe consisting ofat least 8, at least 9, at least 10, at least 11, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, atleast 20, at least 21, at least 22, at least 23, at least 24, or atleast 25 nucleotides, wherein at least a portion of the sequence is atleast 90%, at least 95%, or 100% identical, or at least 90%, at least95%, or 100% complementary, to a region of, the TV 40S ribosomal protein(Tv40Srp) gene. In some embodiments, at least 8, at least 9, at least10, at least 11, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, at least 20, at least 21, at least22, at least 23, at least 24, or at least 25 nucleotides of the FRETprobe are identically present in, or complementary to a region of, theTV 40S ribosomal protein (Tv40Srp) gene. In some embodiments, the FRETprobe has a sequence with one, two or three base mismatches whencompared to the sequence or complement of the TV 40S ribosomal protein(Tv40Srp) gene.

In some embodiments, a kit comprises a polynucleotide discussed above.In some embodiments, a kit comprises at least one primer and/or probediscussed above. In some embodiments, a kit comprises at least onepolymerase, such as a thermostable polymerase. In some embodiments, akit comprises dNTPs. In some embodiments, kits for use in the real timePCR methods described herein comprise one or more target gene-specificFRET probes and/or one or more primers for amplification of targetgenes.

In some embodiments, one or more of the primers and/or probes is“linear”. A “linear” primer refers to a polynucleotide that is a singlestranded molecule, and typically does not comprise a short region of,for example, at least 3, 4 or 5 contiguous nucleotides, which arecomplementary to another region within the same polynucleotide such thatthe primer forms an internal duplex. In some embodiments, the primersfor use in reverse transcription comprise a region of at least 4, suchas at least 5, such as at least 6, such as at least 7 or more contiguousnucleotides at the 3′-end that has a sequence that is complementary toregion of at least 4, such as at least 5, such as at least 6, such as atleast 7 or more contiguous nucleotides at the 5′-end of a target gene.

In some embodiments, a kit comprises one or more pairs of linear primers(a “forward primer” and a “reverse primer”) for amplification of atarget gene. Accordingly, in some embodiments, a first primer comprisesa region of at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19, at least 20, at least 21, at least 22, at least23, at least 24, or at least 25 contiguous nucleotides having a sequencethat is at least 90%, at least 95%, or 100% identical to the sequence ofa region of at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19, at least 20, at least 21, at least 22, at least23, at least 24, or at least 25 contiguous nucleotides at a firstlocation in the target gene. Furthermore, in some embodiments, a secondprimer comprises a region of at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, at least 20, at least 21, atleast 22, at least 23, at least 24, or at least 25 contiguousnucleotides having a sequence that is at least 90%, at least 95%, or100% complementary to the sequence of a region of at least 8, at least9, at least 10, at least 11, at least 12, at least 13, at least 14, atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, at least 21, at least 22, at least 23, at least 24, or at least 25contiguous nucleotides at a second location in the target gene, suchthat a PCR reaction using the two primers results in an ampliconextending from the first location of the target gene to the secondlocation of the target gene.

In some embodiments, the kit comprises at least two, at least three, orat least four sets of primers, each of which is for amplification of adifferent target gene, such as an endogenous control and/or an exogenouscontrol.

In some embodiments, probes and/or primers for use in the compositionsdescribed herein comprise deoxyribonucleotides. In some embodiments,probes and/or primers for use in the compositions described hereincomprise deoxyribonucleotides and one or more nucleotide analogs, suchas LNA analogs or other duplex-stabilizing nucleotide analogs describedabove. In some embodiments, probes and/or primers for use in thecompositions described herein comprise all nucleotide analogs. In someembodiments, the probes and/or primers comprise one or moreduplex-stabilizing nucleotide analogs, such as LNA analogs, in theregion of complementarity.

In some embodiments, the kits for use in real time PCR methods describedherein further comprise reagents for use in the reverse transcriptionand amplification reactions. In some embodiments, the kits compriseenzymes such as heat stable DNA polymerases, such as Taq polymerase. Insome embodiments, the kits further comprise deoxyribonucleotidetriphosphates (dNTP) for use in amplification. In further embodiments,the kits comprise buffers optimized for specific hybridization of theprobes and primers.

A kit generally includes a package with one or more containers holdingthe reagents, as one or more separate compositions or, optionally, as anadmixture where the compatibility of the reagents will allow. The kitcan also include other material(s) that may be desirable from a userstandpoint, such as a buffer(s), a diluent(s), a standard(s), and/or anyother material useful in sample processing, washing, or conducting anyother step of the assay.

Kits preferably include instructions for carrying out one or more of themethods described herein. Instructions included in kits can be affixedto packaging material or can be included as a package insert. While theinstructions are typically written or printed materials they are notlimited to such. Any medium capable of storing such instructions andcommunicating them to an end user is contemplated by this invention.Such media include, but are not limited to, electronic storage media(e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g.,CD ROM), and the like. As used herein, the term “instructions” caninclude the address of an internet site that provides the instructions.

In some embodiments, the kit can comprise the reagents described aboveprovided in one or more GeneXpert® Sample cartridge(s). These cartridgespermit extraction, amplification, and detection to be be carried outwithin this self-contained “laboratory in a cartridge.” (See e.g., U.S.Pat. Nos. 5,958,349, 6,403,037, 6,440,725, 6,783,736, 6,818,185; each ofwhich is herein incorporated by reference in its entirety.) Reagents formeasuring genomic copy number level and detecting a pathogen could beprovided in separate cartridges within a kit or these reagents (adaptedfor multiplex detection) could be provide in a single cartridge.

Any of the kits described here can include, in some embodiments, areceptacle for a urine sample and/or a swab for collecting a urethralswab sample, a vaginal swab sample, or an endocervical swab sample.

The following examples are for illustration purposes only, and are notmeant to be limiting in any way.

5. EXAMPLES 5.1. Example 1: Detection of Trichomonas vaginalis

An assay was designed to detect the gene for the 40S ribosomal protein(Tv40Srp) of Trichomonas vaginalis (TV) by PCR, using the primers andprobe shown in Table 1. In addition to the TV-specific primers andprobe, primers and probe were included to detect a single-copy humangene used as a sample adequacy control (SAC) target. Primers and probewere also included to detect a bacterial gene, which was included in themultiplex reaction as a sample processing control (SPC) target.

TABLE 1 Primer and probe sequences SEQ Amplicon ID SEQ ID oligo nametarget sequence NO NO TV forward Tv40Srp gene GTAACAACCTTGGAGTTCTTCTTAAG1 5 TV reverse Tv40Srp gene ACATCAATCTACAAGACACCACTTGA 2 TV probeTv40Srp gene F1-AGTTTGGCTGCTTAGCTTCGAC-Q1 3

The final primer and probe compositions of the multiplex assay are shownin Table 2.

TABLE 2 Primer and probe concentrations Final conc. Final conc. FinalForw. Rev conc. Target Label Purpose Primer primer Probe Tv40Srp F1 TV 0.3 μM  0.3 μM  0.5 μM detection single-copy F2 SAC 0.35 μM 0.35 μM0.35 μM human gene bacterial gene F2 SPC  0.4 μM  0.4 μM  0.2 μMF1 and F2 are detectably different dyes that can be detected anddistinguished simultaneously in a multiplex reaction. Each probe alsocomprises a quencher (e.g., Q1, above).

Each reaction contained 42-58 mM KCl, 3.5-5.0 mM MgCl₂, 250-350 μMdNTPs, 50 mM Tris, pH 8.6, and 0.01% sodium azide. AptaTaq (0.27-0.37units/μ1; Roche) was used for amplification.

For each sample to be tested, approximately 7 mL of first catch, voidedurine was added to 1 mL of buffer, preferably within 2 hours of samplecollection. Physician-collected endocervical swabs or self-collected (ina clinical setting) vaginal swabs were immediately placed into 2.5 mL ofbuffer.

500 μL of buffered urine or swab sample was loaded into a GeneXpert®cartridge for analysis. The sample was mixed with a lysis reagent torelease nucleic acids. After lysis, the released nucleic acid from thesample was captured on a DNA-binding substrate. The nucleic acid waseluted from the substrate and used to reconstitute the reagents used forreal-time PCR (described above). The reaction cycle used was: 1 minuteat 95° C., followed by up to 40 cycles of 5 seconds at 92.5° C., 20seconds at 68° C. using a GeneXpert® cartridge in a GeneXpert® system.

The results of the assay were interpreted as shown in Table 3. The validrange of Ct values for the TV, SAC, and SPC targets were 9-39.9 Ct.

TABLE 3 Xpert TV assay results and interpretation Result InterpretationTV Trichomonas target DNA is detected. DETECTED The Trichomonas targethas a Ct within the valid range and a fluorescence endpoint above thethreshold setting. SPC-Not applicable. SPC is ignored because theTrichomonas target amplification may compete with this control. SAC-Notapplicable. SAC is ignored because the Trichomonas target amplificationmay compete with this control. PCC-PASS. All probe check results pass.TV NOT Trichomonas target DNA is not detected. SPC meets DETECTEDacceptance criteria. Trichomonas target DNA is not detected. SPC-PASS.SPC has a Ct within the valid range and fluorescence endpoint above thethreshold setting. SAC-PASS. SAC has a Ct within the valid range and afluorescence endpoint above the threshold setting. PCC-PASS. All probecheck results pass. INVALID Presence or absence of Trichomonas targetDNA cannot be determined. Repeat test according to the instructions inSection 11.2, Retest Procedure. SPC-FAIL. SPC Ct is not within validrange and the fluorescence endpoint is below the threshold setting.SAC-PASS. SAC has a Ct within the valid range and fluorescence endpointin the above threshold setting. PCC-PASS. all probe check results pass.Or SPC-PASS. SPC has a Ct within the valid range and fluorescenceendpoint above the threshold setting. SAC-FAIL. SAC Ct is not withinvalid range and fluorescence endpoint is below the threshold setting.PCC-PASS. all probe check results pass. Or SPC-FAIL. SPC Ct is notwithin valid range and fluorescence endpoint is below the thresholdsetting. SAC-FAIL. SAC Ct is not within valid range and fluorescenceendpoint is below the threshold setting. PCC-PASS. All probe checkresults pass. ERROR Presence or absence of Trichomonas target DNA cannotbe determined. Repeat test according to the instructions in Section11.2, Retest Procedure. TRICHOMONAS-NO RESULT SPC-NO RESULT SAC-NORESULT PCC-FAIL.* All or one of the probe check results fail. NO RESULTPresence or absence of Trichomonas target DNA cannot be determined.Repeat test according to the instructions in Section 11.2, RetestProcedure. A NO RESULT indicates that insufficient data were collected.For example, the operator stopped a test that was in progress or a powerfailure occurred. TRICHOMONAS-NO RESULT SPC-NO RESULT SAC-NO RESULTPCC-Not Applicable *If the probe check passed, the error is caused bythe maximum pressure limit exceeding the acceptable range or by a systemcomponent failure.

5.2. Example 2: Clinical Performance

Performance characteristics of the Xpert TV Assay were evaluated at 13institutions in the U.S. Due to the low prevalence of Trichomonasvaginalis and the difficulty in obtaining fresh Trichomonasvaginalis-positive specimens from male subjects, the specimen populationfor this study was supplemented with contrived male urine specimens.

Subjects included consenting asymptomatic and symptomatic, sexuallyactive males and females seen in locations including, but not limitedto: OB/GYN, sexually transmitted disease (STD), teen, public health, andfamily planning clinics.

The study specimens consisted of prospectively collected male urine,female urine, endocervical swabs, and patient-collected vaginal swabs(collected in a clinical setting). Contrived male urine specimens wereincluded to supplement the male sample size.

The Xpert TV Assay performance was compared to an FDA-cleared in vitroqualitative nucleic acid amplification comparator assay that detects theribosomal RNA of Trichomonas vaginalis using a transcription-mediatedassay (APTIMA® Trichomonas vaginalis assay, GenProbe Hologic, San Diego,USA). Samples with discrepant results between the Xpert TV Assay and thecomparator assay were analysed with bi-directional sequencing of aseparate repetitive genomic DNA sequence. See Bandea, et al., Jornal ofClinical Microbiology. 2013, 51(4):1298-1300.

Of the Xpert TV Assays runs performed with eligible specimens, 97.3%(5327/5474) of these specimens were successful on the first attempt. Theremaining 147 gave indeterminate results on the first attempt (91 ERROR,44 INVALID and 12 NO RESULT). One hundred nineteen of the 147 specimensyielded valid results after a single retest; 17 of the specimens wereindeterminate on the second attempt and 11 specimens were not retested.The overall assay success rate was 99.5% (5446/5474).

Results from the Xpert TV Assay were compared to the comparator assay,with bi-directional sequencing of discrepants. Sensitivity andspecificity by gender, specimen type and symptom status are presented inTable 4.

TABLE 4 Xpert TV assay vs. reference NAAT test plus sequencing Sx PrevSensitivity % Specificity % PPV % NPV % Specimen Status n TP FP TN FN %(95 CI) (95 CI) (95 CI) (95 CI) Female PC- Sym 717 63 0 649 5 9.4   92.6100 100   99.2 VS (83.7-97.6) (99.4-100)  (94.3-100) (98.1-99.8) Asym857 50 0 806 1 6.1   98.0 100 100   99.9 (89.6-100)  (99.5-100) (92.9-100) (99.3-100)  All 1574 113 0 1455 6 7.6   95.0 100 100   99.6(89.3-98.1) (99.7-100)  (96.8-100) (99.1-99.8) ES Sym 714 59 0 651 4 8.8  93.7 100 100   99.4 (84.5-98.2) (99.4-100)  (93.9-100) (98.4-99.8)Asym 859 49 0 809 1 5.8   98.0 100 100   99.9 (89.4-99.9) (99.5-100) (92.7-100) (99.3-100)  All 1573 108 0 1460 5 7.2   95.6 100 100   99.7(90.0-98.5) (99.7-100)  (96.6-100) (99.2-99.9) UR Sym 713 60 0 651 2 8.7  96.8 100 100   99.7 (88.8-99.6) (99.4-100)  (94.0-100) (98.9-100) Asym 856 48 0 806 2 5.8   96.0 100 100   99.8 (86.3-99.5) (99.5-100) (92.6-100) (99.1-100)  All 1569 108 0 1457 4 7.1   96.4 100 100   99.7(91.1-99.0) (99.7-100)  (96.6-100) (99.3-99.9) Male OR Sym 125 1 0 124 00.8 100 100 100 100  (2.5-100)  (97.1-100)   (2.5-100) (97.1-100)  Asym411 13 0 398 0 3.2 100 100 100 100 (75.3-100)  (99.1-100)  (75.3-100)(99.1-100)  CS 183 62 19 99 3 NA   95.4   83.9 NA NA (87.1-99.0)(76.0-90.0) All 719 76 19 621 3 NA   96.2   97.0 NA NA (89.3-99.2)(95.4-98.2) a. TP = true positive, FP = false positive, TN = truenegative, FN = false negative, PC-VS = patient-collected vaginal swab,ES = endocervical swab, CS = contrived specimens, UR = urine

5.3. Example 3: Limit of Detection

The analytical sensitivity or limit of detection (LoD) of the Xpert TVAssay was assessed using two Trichomonas vaginalis strains, onemetronidazole susceptible (T. vaginalis ATCC® 30001™), and onemetronidazole resistant (T. vaginalis ATCC® 30238™). Both strains weretested in T. vaginalis-negative pooled male urine (MU) mixed with bufferand T. vaginalis-negative pooled vaginal swab (VS) in buffer

The limit of detection (LoD) was estimated by testing replicates of 20at a minimum of five concentrations for each strain and sample type overthree days. LoDs were estimated by logistic regression. The LoD isdefined as the lowest number of cells/mL that can be reproduciblydistinguished from negative samples with 95% confidence or the lowestconcentration at which 19 of 20 replicates were positive. The study wasperformed with two different lots of Xpert TV reagents and the claimedLoD for each strain is the higher of the two determinations (Table 5).For swab samples in buffer, the limit of detection is 5 cells/mL. Forurine samples in buffer, the limit of detection is 6 cells/mL. Theclaimed LoDs were verified by analyzing at least 20 replicates dilutedto the estimated LoD concentrations.

TABLE 5 Limit of detection of Trichomonas vaginalis using Xpert TV LoDEstimates (Logit) (lower and upper 95% Trichomonas vaginalis confidenceintervals) Verified LoD Verification LoD Claim strain and matrix Lot 1Lot 2 (cells/mL) (Positives/20) (cells/mL) ATCC 30001 in Vaginal Swab3.9 4.2 4.2 20/20 5 (3.0-6.0) (3.3-6.3) ATCC 30238 in Vaginal Swab 4.43.7 4.4 19/20 5 (3.5-6.5) (2.9-5.5) ATCC 30001 in Male Urine 5.8 3.2 5.820/20 6 (4.7-7.9) (2.6-4.8) ATCC 30238 in Male Urine 4.9 4.3 4.9 19/20 5(4.0-6.6) (3.4-6.2)

5.4. Example 4: Assay Reproducibility

A panel of eight specimens with varying concentrations of Trichomonasvaginalis was tested on 12 different days by two different operators, ateach of three sites (8 specimens×1 times/day×12 days×2 operators×3sites). Three lots of Xpert TV Assay were used at each of the 3 testingsites. Xpert TV Assays were performed according to the Xpert TV Assayprocedure. Results are summarized in Table 6.

The reproducibility of the Xpert TV Assay was also evaluated in terms ofthe fluorescence signal expressed in Ct values for each target detected.The mean, standard deviation (SD), and coefficient of variation (CV)between-sites, between-lots, between-days, between-operator, andwithin-assay for each panel member are presented in Table 7.

TABLE 6 Summary of reproducibility results % Total Site 1 Site 2 Site 3Agreement Sample Op 1 Op 2 Site Op 1 Op 2 Site Op 1 Op 2 Site by SampleFS-Neg  100%  100%  100%  100%  100%  100%  100%  100%  100%  100%(24/24) (24/24) (48/48) (24/24) (24/24) (48/48) (24/24) (24/24) (48/48)(144/144) FS-Mod  100%  100%  100%  100%  100%  100%  100%  100%  100% 100% Pos (24/24) (24/24) (48/48) (24/24) (24/24) (48/48) (24/24)(24/24) (48/48) (144/144) FS-Low 62.5% 75.0% 68.8% 70.8% 83.3% 77.1%90.7% 87.5% 79.2% 75.0% Pos (15/24) (18/24) (33/48) (17/24) (20/24)(37/48) (17/24) (21/24) (38/48) (108/144) FS-LoD 91.7%  100% 95.8% 95.8%95.8% 95.8% 95.8%  100% 97.9% 96.5% (22/24) (24/24) (46/48) (23/24)(23/24) (46/48) (23/24) (24/24) (47/48) (139/144) UR-Neg  100%  100% 100%  100%  100%  100%  100%  100%  100%  100% (24/24) (24/24) (48/48)(24/24) (24/24) (48/48) (24/24) (24/24) (48/48) (144/144) UR-Mod  100% 100%  100%  100%  100%  100%  100%  100%  100%  100% Pos (24/24)(24/24) (48/48) (24/24) (24/24) (48/48) (24/24) (24/24) (48/48)(144/144) UR-Low 87.5% 45.8% 66.7% 70.8% 70.8% 70.8% 79.2% 66.7% 72.9%70.1% Pos (31/24) (11/24) (32/48) (17/24) (17/24) (34/48) (19/24)(16/24) (35/48) (101/144) UR-LoD 91.7%  100% 95.8% 95.8% 91.7% 93.8% 100% 91.7% 95.8% 95.1% (22/24) (24/24) (46/48) (23/24) (22/24) (45/48)(24/24) (22/24) (46/48) (137/144) a. FS = female swab matrix; UR = maleurine matrix

TABLE 7 Summary of reproducibility data Between- Between- Between-Between- Within- Assay Site Lot Day Operator Assay Total Channel Mean CVCV CV CV CV CV Sample (Analyte) N^(a) Ct SD (%) SD (%) SD (%) SD (%) SD(%) SD (%) FS-Neg SAC 144 24.61 0 0 0.12 0.5 0.15 0.6 0 0 0.31 1.3 0.371.5 FS-Mod TV 144 35.40 0.09 0.2 0.32 0.9 0.16 0.4 0 0 0.68 1.9 0.77 2.2Pos FS-Low TV 108 38.18 0 0 0 0 0 0 0.52 1.4 0.86 2.3 1.01 2.6 PosFS-LoD TV 139 37.14 0.16 0.4 0.36 1.0 0.21 0.6 0 0 0.92 2.5 1.02 2.7UR-Neg SAC 144 34.10 0.06 0.2 0.20 0.6 0 0 0.17 0.5 0.28 0.8 0.39 1.1UR-Mod TV 144 35.40 0 0 0.37 1.0 0.13 0.4 0.18 0.5 0.65 1.8 0.78 2.2 PosUR-Low TV 101 37.95 0 0 0 0 0.36 1.0 0.52 1.4 0.94 2.5 1.14 3.0 PosUR-LoD TV 137 37.03 0 0 0.33 0.9 0 0 0 0 0.96 2.6 1.01 2.7 ^(a)Resultswith non-zero Ct valuee out of 144

5.5. Example 5: Analytical Inclusivity

The analytical inclusivity of the Xpert TV Assay was evaluated bytesting 17 T. vaginalis strains in triplicate at a concentration nogreater than 3× analytical limit of detection (3×LoD). Strains weretested in T. vaginalis-negative pooled vaginal swab (VS) in buffer, andmale urine (MU) mixed with buffer. See Table 8. Under the conditions ofthis study, all strains reported TV DETECTED results. The Xpert TV assaydemonstrated 100% inclusivity in both sample types.

TABLE 8 Analytical inclusivity panel Results Vaginal Results MaleIsolate ATCC # Isolation Source Swab Urine 30001 Vaginal exudate TVDETECTED TV DETECTED 30184 Vaginal swab TV DETECTED TV DETECTED 30187Endocervical TV DETECTED TV DETECTED swab 30188 Vagina TV DETECTED TVDETECTED 30236 Endocervical TV DETECTED TV DETECTED swab 30240 Vaginalpool TV DETECTED TV DETECTED 30245 Vaginal and TV DETECTED TV DETECTEDEndocervical material 30247 Vagina TV DETECTED TV DETECTED 50138 humanTV DETECTED TV DETECTED 50139 human TV DETECTED TV DETECTED 50141 humanTV DETECTED TV DETECTED 50143 human TV DETECTED TV DETECTED 50147 humanTV DETECTED TV DETECTED 50167 Vagina TV DETECTED TV DETECTED 50183Prostatic fluid TV DETECTED TV DETECTED PRA-95 Vaginal TV DETECTED TVDETECTED exudate PRA-98 human TV DETECTED TV DETECTED

5.6. Example 6: Analytical Specificity

A panel of 47 organisms, including bacteria, fungi, and viruses commonlyfound in the urogenital tract, as well as other closely relatedprotozoans to Trichomonas were tested with the Xpert TV Assay. Eachbacterial or fungal strain was tested at 1×10⁷ cfu/mL or greater.Strains which did not produce countable colonies were diluted to 0.5McFarland units, approximately equivalent to 1.5×108 cfu per mL for E.coli. Viral strains were purchased as heat inactivated stocks fromZeptoMetrix Corp. and tested at 1×10⁶ U/mL or 10⁶ genomes/mL. Protozoanswere cultured in growth media, visually enumerated by light microscopyand tested at 1×10⁶ cells/mL. Tests were performed in triplicate. Theorganisms tested and the Xpert TV assay results are listed in Table 9.

One organism, Trichomonas tenax, reported a TV DETECTED result with theXpert TV assay. Under the conditions of this study, the analyticalspecificity of the Xpert TV Assay was 98%.

TABLE 9 Analytical specificity panel Species Strain ID Test ResultAcinetobacter Iwoffi ATCC 17925 TV NOT DETECTED Actinomyces israeliiATCC 12102 TV NOT DETECTED Atopobium vaginae ATCC BAA-55 TV NOT DETECTEDBacteroides fragilis ATCC 25285 TV NOT DETECTED Bacteroides ureolyticusATCC 33387 TV NOT DETECTED Bifidobacterium adolescentis ATCC 15703 TVNOT DETECTED Campylobacter jejuni ATCC 33560 TV NOT DETECTED Candidaalbicans ATCC 14053 TV NOT DETECTED Candida glabrata ATCC 90030 TV NOTDETECTED Candida parapsilosis ATCC 90018 TV NOT DETECTED Candidatropicalis ATCC 13803 TV NOT DETECTED Chlamydia trachomatis ATCC VR-885TV NOT DETECTED Clostridium difficile ATCC 43594 TV NOT DETECTEDClostridium perfringens ATCC 13124 TV NOT DETECTED Corynebacteriumgenitalium ATCC 33031 TV NOT DETECTED Cryptococcus neoformans ATCC 32045TV NOT DETECTED Cytomegalovirus ZeptoMetrix TV NOT DETECTED 0810003CFEnterobacter aerogenes ATCC 51697 TV NOT DETECTED Enterococcus feacalisATCC 19433 TV NOT DETECTED Escherichia coil ATCC 24922 TV NOT DETECTEDFusobacterium nucleatum ATCC 31647 TV NOT DETECTED Gardnerella vaginalisATCC 49145 TV NOT DETECTED Haemophilus ducreyi ATCC 33940 TV NOTDETECTED Herpes simplex virus I ZeptoMetrix TV NOT DETECTED 0810005CFHerpes simplex virus II ZeptoMetrix TV NOT DETECTED 0810006CF HIV-1ZeptoMetrix TV NOT DETECTED 0801032CF HPV 16 (Caski) ZeptoMetrix TV NOTDETECTED 0810232 Klebsiella oxytoca ATCC 43165 TV NOT DETECTEDLactobacillus acidophilus ATCC 314 TV NOT DETECTED Lactobacillusjensenii ATCC 25258 TV NOT DETECTED Lactobacillus vaginalis ATCC 49540TV NOT DETECTED Listeria monocytogenes ATCC 15313 TV NOT DETECTEDMobiluncus curtisil ATCC 35241 TV NOT DETECTED Mycoplasma hominis ATCC23114 TV NOT DETECTED Neisseria gonorrhoeae ATCC 35201 TV NOT DETECTEDPentatrichomonas hominis ATCC 30000 TV NOT DETECTED Peptostreptococcusanaerobius ATCC 49031 TV NOT DETECTED Prevotella bivia ATCC 29303 TV NOTDETECTED Propionibacterium acnes ATCC 6919 TV NOT DETECTED Proteusmirabilis ATCC 25933 TV NOT DETECTED Pseudomonas aeruginosa ATCC 35554TV NOT DETECTED Staphylococcus aureus ATCC 700699 TV NOT DETECTEDStaphylococcus epidermidis ATCC 14990 TV NOT DETECTED Streptococcusagalactiae ATCC 13813 TV NOT DETECTED Streptococcus pyogenes ATCC 19615TV NOT DETECTED Trichomonas tenax ATCC 30207 TV DETECTED Ureaplasmaurealyticum ATCC 27618 TV NOT DETECTED

5.7. Example 7: Interfering Substances

In a non-clinical study, potentially interfering endogenous andexogenous substances that may be within the urogenital tract and presentin endocervical and vaginal swab or first catch urine samples wereevaluated with the Xpert TV Assay.

Substances were individually diluted into a pooled negative vaginal swabmatrix and a pooled negative male urine matrix. The substances were alsotested in the same matrices spiked with T. vaginalis cells at no greaterthan three times the limit of detection for the respective sample type.Eight replicates of each set of negative and positive samples weretested with the Xpert TV assay and compared to the results obtained in acontrol of the same sample without the potential interfering substanceadded. The substances and test concentrations are listed in Table 10 andTable 11.

Under the conditions of the study, in tests with the substances dilutedinto negative urine matrix no invalid results were reported; all testsreported TV NOT DETECTED as expected. Assay interference was observed intests with blood at 0.75% v/v and azithromycin at 1.8 mg/mL diluted intopositive urine matrix. False negative results were not reported fortests with blood at 0.5% v/v and azithromycin at 1 mg/mL.

Under the conditions of the study, in tests with the substances dilutedinto pooled negative swab matrix no invalid results were reported; alltests reported TV NOT DETECTED as expected.

In testing of substances diluted into pooled positive swab matrix, nofalse negative TV results were reported. Testing with all the substancesreported TV DETECTED results as expected.

TABLE 10 Potentially interfering substances in urine samplesConcentration Class/Substance Active Ingredient Tested Blood Blood 0.3%v/v Seminal Fluid Seminal Fluid 5.0% v/v Mucus Mucin 0.8% w/v Analgesics& Acetylsalicylic Acid 500 mg 8 mg/mL Antibiotics Acetaminophen 3.2mg/mL Azithromycin 1.0 mg/mL Doxycycline 0.5 mg/mL OTC Deodorant &PEG-20; PEG-32; 0.25% w/v Powders PEG-20 Stearate Nanoxynol-9 0.25% w/vAlbumin BSA 10 mg/ml Glucose Glucose 10 mg/ml Bilirubin Bilirubin 1mg/ml Acidic Urine (pH 4.0) Urine + N-Acetyl-L-Cysteine pH 4.0 AlkalineUrine (pH 9.0) Urine + Ammonium Citrate pH 9.0 Leukocytes Leukocytes 10⁶cells/mL Intravaginal Hormones Progesterone; Estradiol 7 mg/mLProgesterone + 0.07 mg/mL Beta Estradiol

TABLE 11 Potentially interfering substances in swab samplesClass/Substance Active Ingredient Concentration Tested Blood Blood  1.0%v/v Seminal Fluid Seminal Fluid  5.0% v/v Mucus Mucin  0.8% w/v Over thecounter (OTC) Benzocaine 5%; 0.25% w/v Vaginal Products; Resorcinol 2%Contraceptives; Vaginal Clotrimazole 2% 0.25% w/v treatments MiconazoleNitrate 2% 0.25% w/v Tioconazole 0.25% w/v 5% w/w Aciclovir 0.25% w/vGlycerin, Propylene 0.25% w/v glycol Glycerin; Carbomer 0.12% w/vGlycerin, Hydroxyethyl 0.25% w/v cellulose Goldenseal 3X HPUS; 0.25% w/vKreosotum 12X HPUS Povidone-iodine 10% 0.25% v/v Nonoxynol-9 12.5% 0.25%w/v Hemerrhoidal Cream Glycerin 14%; 0.25% w/v Pramoxine HCl 1%Leukocytes Leukocytes 10⁶ cells/mL Intravaginal Hormones Progesterone;Estradiol 7 mg/mL Progesterone + 0.07 mg/mL Beta Estradiol

5.8. Example 8: Carry-Over Contamination

The study consisted of repeated tests of a TV-negative vaginal swab poolin buffer sample processed within the same GeneXpert module immediatelyfollowing a high (10⁶ cells/mL) TV positive vaginal swab pool in buffersample. The study consisted of a TV-negative vaginal swab pool in buffersample processed within the same GeneXpert module immediately followedby a high (10⁶ cells/mL) TV positive vaginal swab pool in buffer sample.This testing scheme was repeated a further 20 times on two GeneXpertmodules for a total of 82 runs resulting in 40 positive and 42 negativesamples. All 40 positive samples were correctly reported as TV DETECTEDand all 42 negative samples were correctly reported as TV NOT DETECTED.

5.9. Example 9: Alternate Primers and Probes Tested to Detect TV

To develop the TV assay described herein, four different forward primer,two different reverse primers, and two different probes for detectingthe TV 40S ribosomal protein (Tv40Srp) gene were tested for sensitivityand specificity (e.g., cross-reactivity with other species) in theassay. Table 12 shows the tested primers and probes.

TABLE 12 Alternate primer and probe sequences SEQ ID oligo name NOsequence Results TV forward  1 GTAACAACCTTGGAGTTCTTCTTAAG Final designTV reverse  2 ACATCAATCTACAAGACACCACTTGA Final design TV probe  3F1-AGTTTGGCTGCTTAGCTTCGAC-Q1 Final design TV forward ALT1  6GAGTTCTTCTTAAGCTGAACAC FW design 2 TV forward ALT2  7GAGTTCTTCTTGAGCTGAACAC FW design 2 with different SNP at position 12TV forward ALT3  8 AACAACCTTGGAGTTCTTCTTA FW design 3 TV reverse ALT1  9ATCTACAAGACACCACTTGA RV design 2 TV probe ALT1 10F1-AGTTTGGCTGCTTGGCTTCGAC-Q1 PR design 2 with differentSNP at position 14

It was found that TV forward ALT1 cross-reacted with Pentatric homonashominis (Pth), another closely related trichomonad found in human gut.When TV forward ALT1 was used in an assay with 1000 copies of TV and anassay with 500,000 copies of Pth, TV was detected with a Ct of 30.7 andPth was detected with a Ct of 26.3. TV forward ALT2 was less sensitivethan the final design, detecting TV with a higher Ct value of 31.5.Similarly, TV forward ALT3 was less sensitive than the final design,also detecting TV with a higher Ct value. TV reverse ALT1 also resultedin a less sensitive assay, detecting TV with a higher Ct value. Finally,TV probe ALT1 was less sensitive and less consistent than the finaldesign.

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that changes can be made without departing fromthe spirit and scope of the invention(s).

TABLE OF CERTAIN SEQUENCES SEQ ID NO Description Sequence 4 Trichomonasggccggcctt tctgatgggt aagtctaaag cttgcggtcg tctcgctgct vaginalis 40Scgtaaactcc gtcttgcaca caagtccaac ttgtgggctt ccaacgcata ribosomal proteinccgccgttcc cttggtacat caatctacaa gacaccactt gagggtacat (Tv40Srp) genecaatggcatc tggcatcgtc gtcggcaagg tcgctgtcga agccaagcagccaaactctg ctattcgtaa agctgtccgt gttcagctta agaagaactctaaggttgtc acagctttcg ttccacgcga tggttccctc cgtcttattgatgataacga ccgtgttctt attgccggta tgggtcgttc tggccgttctgtcggtgacc ttccaggatg ccgtttcaaa gttatcaagg tcgctggtttctccctcctt gctctttggc tcggcaagaa ggagaagccg cgcagctaaataaatactct tgggtttacc ggtaaataaa aacatatatt acgaaataca aatattat 5Tv40SrpACATCAATCT ACAAGACACC ACTTGAAGGC ACCTCAATGG CCTCCGGCAT TGTTGTCGGCampliconAAAGTTGCTG TCGAAGCTAA GCAGCCAAAC TCCGCTATTC GTAAAGCAGT TCGTGTTCAGCTTAAGAAGA ACTCTAAAGT TGTTAC

What is claimed is:
 1. A composition comprising a set of primers and aprobe for detecting a Trichomonas vaginalis 40S ribosomal protein(Tv40Srp) gene or RNA, wherein the Tv40Srp gene comprises the sequenceof SEQ ID NO: 4, and wherein the set of primers comprises a first primercomprising a region of at least 8 contiguous nucleotides having asequence that is at least 90% identical to a region of at least 8contiguous nucleotides of SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 7, orSEQ ID NO: 8; and a second primer comprising a region of at least 8contiguous nucleotides having a sequence that is at least 90%complementary to a region of at least 8 contiguous nucleotides of SEQ IDNO: 2 or SEQ ID NO: 9; wherein the probe is capable of selectivelyhybridizing to a Tv40Srp amplicon produced by the first primer andsecond primer, and wherein the probe comprises a region of at least 8contiguous nucleotides having a sequence that is at least 90% identicalor complementary to a region of at least 8 contiguous nucleotides of SEQID NO: 3 or SEQ ID NO: 10 and comprises a detectable label.
 2. Thecomposition of claim 1, wherein the first primer comprises a region ofat least 8 contiguous nucleotides having a sequence that is identical toa region of at least 8 contiguous nucleotides of SEQ ID NO: 1 and thesecond primer comprises a region of at least 8 contiguous nucleotideshaving a sequence that is identical to a region of at least 8 contiguousnucleotides of SEQ ID NO:
 2. 3. The composition of claim 1, wherein theprobe comprises a region of nucleotides having a sequence that is atleast 90%, at least 95%, or 100% identical or complementary to a regionof at least 15, at least 16, at least 17, at least 18, at least 19, atleast 20, at least 21, or at least 22 contiguous nucleotides of SEQ IDNO: 3 or SEQ ID NO:
 10. 4. The composition of claim 1, wherein the probecomprises a region of at least 8 contiguous nucleotides having asequence that is identical or complementary to a region of at least 8contiguous nucleotides of SEQ ID NO: 3 or SEQ ID NO:
 10. 5. Thecomposition of claim 1, wherein the first primer comprises a region ofat least 8 contiguous nucleotides having a sequence that is identical toa region of at least 8 contiguous nucleotides of SEQ ID NO: 1, and thesecond primer comprises a region of at least 8 contiguous nucleotideshaving a sequence that is identical to a region of at least 8 contiguousnucleotides of SEQ ID NO: 2, and wherein the probe comprises a region ofat least 8 contiguous nucleotides having a sequence that is identical orcomplementary to a region of at least 8 contiguous nucleotides of SEQ IDNO:
 3. 6. The composition of claim 1, wherein the composition is alyophilized composition or is in solution.
 7. A kit comprising a set ofprimers and a probe for detecting a Trichomonas vaginalis 40S ribosomalprotein (Tv40Srp) gene or RNA, wherein the Tv40Srp gene comprises thesequence of SEQ ID NO: 4, and wherein the set of primers comprises afirst primer comprising a region of at least 8 contiguous nucleotideshaving a sequence that is at least 90% identical to a region of at least8 contiguous nucleotides of SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 7, orSEQ ID NO: 8; and a second primer comprising a region of at least 8contiguous nucleotides having a sequence that is at least 90%complementary to a region of at least 8 contiguous nucleotides of SEQ IDNO: 2 or SEQ ID NO: 9, wherein the probe is capable of selectivelyhybridizing to a Tv40Srp amplicon produced by the first primer andsecond primer, and wherein the probe comprises a region of at least 8contiguous nucleotides that is at least 90% identical or complementaryto a region of at least 8 contiguous nucleotides of SEQ ID NO: 3 or SEQID NO: 10 and comprises a detectable label.
 8. The kit of claim 7,wherein the kit comprises a primer pair for detecting an endogenouscontrol and/or a primer pair for detecting an exogenous control, whereinthe primer pairs for detecting Tv40Srp and the controls are each in thesame or different compositions in the kit.
 9. The kit of claim 8,wherein the endogenous control is a sample adequacy control.
 10. The kitof claim 8, wherein the endogenous control is selected from HMBS, GAPDH,beta actin, and beta globin.
 11. The kit of claim 8, wherein theexogenous control is a sample processing control.
 12. The kit of claim8, wherein the exogenous control is a bacterial gene.
 13. The kit ofclaim 7, wherein the first primer comprises a region of at least 8contiguous nucleotides having a sequence that is identical to a regionof at least 8 contiguous nucleotides of SEQ ID NO: 1, and the secondprimer comprises a region of at least 8 contiguous nucleotides having asequence that is identical to a region of at least 8 contiguousnucleotides of SEQ ID NO:
 2. 14. The kit of claim 8, wherein the kitfurther comprises a second probe capable of selectively hybridizing toan endogenous control amplicon, and/or a third probe capable ofselectively hybridizing to an exogenous control amplicon, wherein theprobes are in the same or different composition from one or more of theprimer pairs.
 15. The kit of claim 7, wherein the probe comprises aregion of at least 8 nucleotides that is identical or complementary to aregion of at least 8 contiguous nucleotides of SEQ ID NO: 3 or SEQ IDNO:
 10. 16. The kit of claim 7, wherein the probe comprises the sequenceof SEQ ID NO: 3 or SEQ ID NO:
 10. 17. A kit comprising a set of primersand a probe for detecting a Trichomonas vaginalis 40S ribosomal protein(Tv40Srp) gene or RNA, wherein the Tv40Srp gene comprises the sequenceof SEQ ID NO: 4, and wherein the set of primers comprises a first primercomprising a region of at least 8 contiguous nucleotides having asequence that is at least 90% identical to a region of at least 8contiguous nucleotides of SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 7, orSEQ ID NO: 8; and a second primer comprising a region of at least 8nucleotides having a sequence that is at least 90% complementary to aregion of at least 8 contiguous nucleotides of SEQ ID NO: 2 or SEQ IDNO: 9, wherein the probe is capable of selectively hybridizing to aTv40Srp amplicon produced by the first primer and second primer, whereinthe probe comprises the sequence of SEQ ID NO: 3 or SEQ ID NO: 10 andcomprises a detectable label.
 18. The composition of claim 1, whereinthe first primer comprises the sequence of SEQ ID NO: 1, SEQ ID NO: 6,SEQ ID NO: 7, or SEQ ID NO: 8 and the second primer comprises thesequence of SEQ ID NO: 2 or SEQ ID NO:
 9. 19. The composition of claim1, wherein the first primer comprises the sequence of SEQ ID NO: 1 andthe second primer comprises the sequence of SEQ ID NO:
 2. 20. The kit ofclaim 7, wherein the first primer comprises the sequence of SEQ ID NO:1, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 and the second primercomprises the sequence of SEQ ID NO: 2 or SEQ ID NO:
 9. 21. The kit ofclaim 7, wherein the first primer comprises the sequence of SEQ ID NO: 1and the second primer comprises the sequence of SEQ ID NO: 2.